Programmable nuclease improvements and compositions and methods for nucleic acid amplification and detection

ABSTRACT

Disclosed herein are compositions, kits, and methods related to improved Cas activity. Through compositions and kits disclosed herein and practice of methods disclosed herein, one attains improved Cas activity such as Cas12 activity relative to Cas proteins in the art such as LbCas12. Further described herein are methods to detect target nucleic acid using a programmable nuclease system. Often, the target nucleic acids are present in at low frequency in the sample. Provided herein are methods for enriching these target nucleic acids for detection. Also described herein are methods to insert a PAM sequence into a target sequence of interest for use in a detection comprising a programmable nuclease.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.17/037,620 filed Sep. 29, 2020, which is a continuation of PCTInternational Application No. PCT/US2020/012276, filed Jan. 3, 2020,which claims priority to and the benefit from U.S. ProvisionalApplication Nos. 62/788,704 filed Jan. 4, 2019; 62/788,706 filed Jan. 4,2019; 62/795,463 filed Jan. 22, 2019; 62/863,166 filed Jun. 18, 2019;62/881,801 filed Aug. 1, 2019; 62/894,515 filed Aug. 30, 2019;62/944,933 filed Dec. 6, 2019; and 62/944,939 filed Dec. 6, 2019, theentire contents of each of which are herein incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 12, 2020, isnamed 53694-724_601_SL.txt and is 1,291,429 bytes in size.

BACKGROUND

CRISPR/Cas-based diagnostics can be very useful for early detection ofnucleic acids associated with disease, however, there still exists aneed for formulations of Cas proteins and reagents that exhibit optimalactivity in diagnostic assays.

Assaying of a target nucleic acid comprising a mutation can bedifficult, especially in the presence of a nucleic acid comprising avariant of the mutation because the mutation is the only differencebetween the sequences of these nucleic acids. This becomes moredifficult when the mutation is a single nucleotide mutation.Additionally, it is often difficult to assay for the target nucleic acidcomprising the mutation when the sample comprising the target nucleicacid also comprises more of the nucleic acid comprising the variant ofthe mutation than the target nucleic acid comprising the mutation.Therefore, there is a need for enhanced detection of a target nucleicacid with a mutation in a sample also comprising a nucleic acidcomprising a variant of the mutation.

There are many target nucleic acids of interest that do not encode forthe PAM sequence. However, a target nucleic acid is may need a PAMsequence for binding and trans cleavage activation of some programmablenucleases complexed with a guide nucleic acid. Therefore, there is aneed for strategies to allow for binding and trans cleavage activationof the programmable nucleases complexed with a guide nucleic acid usingany target nucleic sequence of interest.

SUMMARY

In various aspects, the present disclosure provides a compositioncomprising a programmable nuclease having at least 60% sequence identityto SEQ ID NO: 11 and a non-naturally occurring guide nucleic acid.

In some aspects, the programmable nuclease comprises a turnover rate ofat least about 0.1 cleaved detector nucleic acid molecules per minute.In some aspects, the programmable nuclease recognizes a protospaceradjacent motif of YYN.

In various aspects, the present disclosure provides a compositioncomprising programmable nuclease having a turnover rate of at leastabout 0.1 cleaved detector nucleic acid molecules per minute and anon-naturally occurring guide nucleic acid.

In some aspects, the programmable nuclease recognizes a protospaceradjacent motif of YYN.

In various aspects, the present disclosure provides a compositioncomprising a non-naturally occurring guide nucleic acid and aprogrammable nuclease, wherein the programmable nuclease comprises aturnover rate of at least about 0.1 cleaved detector nucleic acidmolecules per minute and recognizes a protospacer adjacent motif of YYN.

In some aspects, the programmable nuclease is a Type V programmablenuclease. In some aspects, the programmable nuclease is a Cas12nuclease. In some aspects, the programmable nuclease comprises threepartial RuvC domains. In some aspects, the programmable nucleasecomprises a RuvC-I subdomain, a RuvC-II subdomain, and a RuvC-IIIsubdomain.

In some aspects, the programmable nuclease has at least 60% sequenceidentity to SEQ ID NO: 11. In some aspects, the programmable nucleasehas at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 92%, at least 95%, at least 97%, or at least 99% sequenceidentity to SEQ ID NO: 11. In some aspects, the programmable nuclease isSEQ ID NO: 11.

In some aspects, the Y is a C or T nucleotide. In some aspects, the N isany nucleotide. In some aspects, the composition further comprises abuffer. In some aspects, the buffer comprises a buffering agent, a salt,a crowding agent, a detergent, or any combination thereof.

In some aspects, the buffering agent is at a concentration of from 5 mMto 100 mM. In some aspects, the buffering agent is at a concentration offrom 10 mM to 40 mM. In some aspects, the buffering agent is at aconcentration of about 20 mM. In some aspects, the salt is from 5 mM to100 mM. In some aspects, the salt is from 5 mM to 10 mM. In someaspects, the crowding agent is from 0.5% (v/v) to 2% (v/v). In someaspects, the crowding agent is about 1% (v/v). In some aspects, thedetergent is about 2% (v/v) or less In some aspects, the detergent isabout 0.00016% (v/v). In some aspects, the buffering agent is HEPES. Insome aspects, the salt is potassium acetate, magnesium acetate, sodiumchloride, magnesium chloride, or any combination thereof.

In some aspects, the crowding agent is glycerol. In some aspects, thedetergent is Tween, Triton-X, or any combination thereof. In someaspects, a pH of the composition is from 7 to 8. In some aspects, a pHof the composition is 7.5. In some aspects, the composition is at atemperature of from 25° C. to 45° C. In some aspects, the programmablenuclease exhibits catalytic activity at a temperature of from 25° C. to45° C. In some aspects, the programmable nuclease exhibits catalyticactivity after heating the composition to a temperature of greater than45° C. and restoring the temperature to from 25° C. to 45° C.

In various aspects, the present disclosure provides a method of assayingfor a segment of a target nucleic acid in a sample, the methodcomprising: contacting the sample to: a detector nucleic acid; and anyof the above described compositions, wherein the guide nucleic acidhybridizes to a segment of the target nucleic acid; and assaying for asignal produced by cleavage of the detector nucleic acid.

In various aspects, the present disclosure provides a method of assayingfor a segment of a target nucleic acid in a sample from a subjectcomprising: contacting the sample comprising a population of nucleicacids to: a guide nucleic acid that hybridizes to the segment of thetarget nucleic acid; a detector nucleic acid; and a Cas12 nuclease thatcleaves the detector nucleic acid upon hybridization of the guidenucleic acid to the segment of the target nucleic acid; and assaying fora signal produced by cleavage of the detector nucleic acid, wherein thesignal is at least two-fold greater when the segment of the targetnucleic acid is present in the sample than the signal when the samplelacks the segment of the target nucleic acid and wherein the subject hasa disease when the segment of the target nucleic acid is present.

In some aspects, the method further comprising administering a treatmentfor the disease.

In various aspects, the present disclosure provides a method of assayingfor a segment of a target nucleic acid comprising: contacting a samplecomprising a population of nucleic acids, wherein the populationcomprises at least one nucleic acid comprising a segment having lessthan 100% sequence identity to the segment of the target nucleic acidand having no less than 50% sequence identity to the segment of thetarget nucleic acid to: a guide nucleic acid that hybridizes to thesegment of the target nucleic acid; a detector nucleic acid; and a Cas12nuclease that cleaves the detector nucleic acid upon hybridization ofthe guide nucleic acid to the segment of the target nucleic acid; andassaying for a signal produced by cleavage of the detector nucleic acid,wherein the signal is at least two-fold greater when the segment of thetarget nucleic acid is present in the sample than the signal when thesample lacks the segment of the target nucleic acid.

In some aspects, the segment of the at least one nucleic acid comprisesat least two base mutations compared to the segment of the targetnucleic acid. In some aspects, the segment of the at least one nucleicacid comprises from one to ten base mutations compared to the segment ofthe target nucleic acid. In some aspects, the segment of the at leastone nucleic acid comprises one base mutation compared to the segment ofthe target nucleic acid. In some aspects, the signal is from two-fold to20-fold greater when the segment of the target nucleic acid is presentin the sample than the signal when the sample lacks the segment of thetarget nucleic acid. In some aspects, the signal is from two-fold to10-fold greater when the segment of the target nucleic acid is presentin the sample than the signal when the sample lacks the segment of thetarget nucleic acid. In some aspects, the signal is from five-fold to10-fold greater when the segment of the target nucleic acid is presentin the sample than the signal when the sample lacks the segment of thetarget nucleic acid.

In some aspects, the guide nucleic acid is reverse complementary to thesegment of the target nucleic acid In some aspects, the guide nucleicacid and the second guide nucleic acid lack synthetic mismatches. Insome aspects, the guide nucleic acid is at least 10 bases. In someaspects, the guide nucleic acid is from 10 to 50 bases. In some aspects,the guide nucleic acid is at least 25 bases. In some aspects, the targetnucleic acid is in the population of nucleic acids at a minor allelefrequency of 10% or less. In some aspects, the target nucleic acid is inthe population of nucleic acids at a minor allele frequency of from 0.1%to 10%. In some aspects, the target nucleic acid is in the population ofnucleic acids at a minor allele frequency of from 0.1% to 5%. In someaspects, the target nucleic acid is in the population of nucleic acidsat a minor allele frequency of from 0.1% to 1%.

In some aspects, the Cas12 nuclease is Cas12a, Cas12b, Cas12c, CasY, orCas12e. In some aspects, the Cas12 nuclease is Cas12a. In some aspects,the Cas12 nuclease has at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 92%, at least 95%, at least 97%, atleast 99%, or 100% sequence identity to any one of SEQ ID NO: 1-SEQ IDNO: 11, SEQ ID NO: 282, or SEQ ID NO: 571-SEQ ID NO: 602. In someaspects, the Cas12 nuclease has at least 60% sequence identity to SEQ IDNO: 11.

In some aspects, the contacting is carried out in a buffer comprising abuffering agent, a salt, a crowding agent, a detergent, a reducingagent, a competitor, or any combination thereof. In some aspects, thebuffering agent is at a concentration of from 5 mM to 100 mM. In someaspects, the buffering agent is at a concentration of from 10 mM to 30mM. In some aspects, the salt is from 5 mM to 100 mM. In some aspects,the salt is from 5 mM to 10 mM. In some aspects, the crowding agent isfrom 0.5% (v/v) to 10% (v/v). In some aspects, the crowding agent isfrom 1% (v/v) to 5% (v/v). In some aspects, the detergent is at 2% (v/v)or less In some aspects, the reducing agent is from 0.01 mM to 100 mM.In some aspects, the reducing agent is from 0.1 mM to 10 mM. In someaspects, the reducing agent is from 0.5 mM to 2 mM. In some aspects, thecompetitor is from 1 ug/ml to 100 ug/ml. In some aspects, the competitoris from 40 ug/ml to 60 ug/ml.

In some aspects, the buffering agent is HEPES, Tris, or any combinationthereof. In some aspects, the salt is potassium acetate, magnesiumacetate, sodium chloride, magnesium chloride, or any combinationthereof. In some aspects, the crowding agent is glycerol. In someaspects, the detergent is Tween, Triton-X, or any combination thereof.In some aspects, the reducing agent is DTT. In some aspects, thecompetitor is heparin. In some aspects, a pH of the composition is from7 to 8.

In some aspects, the method further comprises amplifying the targetnucleic acid before the contacting. In some aspects, the amplifying thetarget nucleic acid before the contacting comprises using a blockingprimer. In some aspects, the target nucleic acid segment comprises asingle nucleotide mutation. In some aspects, the blocking primer bindsto a nucleic acid comprising encoding the wild type sequence of thetarget nucleic acid segment. In some aspects, the amplifying comprisesCOLD-PCR. In some aspects, the COLD-PCR comprises full COLD-PCR. In someaspects, the COLD-PCR comprises fast COLD-PCR. In some aspects, theamplifying comprises fast COLD-PCR. In some aspects, the amplifyingcomprises allele-specific PCR. In some aspects, the amplifying furthercomprises COLD-PCR.

In various aspects, the present disclosure provides a compositioncomprising a programmable nuclease and a buffer, wherein the buffercomprises a salt at less than about 110 mM and wherein the buffercomprises a pH of from 7 to 8.

In some aspects, the salt is from 1 mM to 110 mM. In some aspects, thesalt is from 1 mM to 60 mM. In some aspects, the salt is from 1 mM to 10mM. In some aspects, the salt is at about 105 mM. In some aspects, thesalt is at about 55 mM. In some aspects, the salt is at about 7 mM. Insome aspects, the salt comprises potassium acetate, magnesium acetate,sodium chloride, magnesium chloride, potassium chloride, or anycombination thereof. In some aspects, the salt comprises potassiumacetate and magnesium acetate. In some aspects, the salt comprisessodium chloride and magnesium chloride. In some aspects, the saltcomprises potassium chloride and magnesium chloride.

In some aspects, the pH comprises about 7.5. In some aspects, the pHcomprises about 8. In some aspects, the buffer comprises a crowdingagent or a competitor. In some aspects, the crowding agent is presentfrom 1% (v/v) to 10% (v/v). In some aspects, the crowding agent or thecompetitor is present from 1% (v/v) to 5% (v/v). In some aspects, thecrowding agent or the competitor is present at about 5% (v/v). In someaspects, the crowding agent or the competitor is present at about 1%(v/v). In some aspects, the crowding agent or the competitor is presentfrom 1 ug/mL to 100 ug/ml. In some aspects, the crowding agent or thecompetitor is present from 30 ug/ml to 70 ug/ml. In some aspects, thecrowding agent or the competitor is present at about 50 ug/ml. In someaspects, the crowding agent or the competitor is present from 1 mM to 50mM. In some aspects, the crowding agent or the competitor is presentfrom 10 mM to 30 mM. In some aspects, the crowding agent or thecompetitor is present at about 20 mM.

In some aspects, the crowding agent or the competitor is selected fromthe group consisting of: glycerol, heparin, bovine serum albumin,imidazole, and any combination thereof. In some aspects, the crowdingagent or the competitor comprises glycerol. In some aspects, thecrowding agent or the competitor comprises glycerol and heparin. In someaspects, the crowding agent or the competitor comprises glycerol, bovineserum albumin, and imidazole. In some aspects, the buffer comprises abuffering agent. In some aspects, the buffering agent is present from 1mM to 50 mM. In some aspects, the buffering agent is present from 1 mMto 30 mM. In some aspects, the buffering agent is present at about 20mM. In some aspects, the buffering agent is HEPES. In some aspects, thebuffering agent is Tris.

In some aspects, the buffer comprises a detergent. In some aspects, thedetergent is present from 0.00001% (v/v) to 0.1% (v/v). In some aspects,the detergent is present from 0.00001% (v/v) to 0.01% (v/v). In someaspects, the detergent is at about 0.00016% (v/v). In some aspects, thedetergent is at about 0.01% (v/v). In some aspects, the detergent isTriton-X. In some aspects, the detergent is IGEPAL CA-630. In someaspects, the buffer comprises a reducing agent. In some aspects, thereducing agent is present from 0.01 mM to 100 mM. In some aspects, thereducing agent is present from 0.1 mM to 10 mM. In some aspects, thereducing agent is present at about 1 mM. In some aspects, the reducingagent is DTT.

In some aspects, the programmable nuclease comprises a RuvC domain. Insome aspects, the programmable nuclease comprises a Type V Cas protein.In some aspects, the programmable nuclease is a Cas12 protein. In someaspects, the Cas12 protein is Cas12a, Cas12b, Cas12c, CasY, or Cas12e.In some aspects, the programmable nuclease has at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 92%, at least95%, at least 97%, at least 99%, or 100% sequence identity to any one ofSEQ ID NO: 1-SEQ ID NO: 11, SEQ ID NO: 282, or SEQ ID NO: 571-SEQ ID NO:602. In some aspects, the programmable nuclease has at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 95%, at least 97%, at least 99%, or 100% sequence identity to SEQID NO: 1. In some aspects, the programmable nuclease has at least 60%,at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 95%, at least 97%, at least 99%, or 100% sequence identity to SEQID NO: 11. In some aspects, the programmable nuclease comprises at leasttwo HEPN domains.

In some aspects, the programmable nuclease is a Type VI Cas protein. Insome aspects, the programmable nuclease is a Cas13 protein. In someaspects, the Cas13 protein is Cas13a, Cas13b, Cas13c, Cas13d, or Cas13e.In some aspects, the programmable nuclease has at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 92%, at least95%, at least 97%, at least 99%, or 100% sequence identity to any one ofSEQ ID NO: 103-SEQ ID NO: 137. In some aspects, the programmablenuclease has at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 92%, at least 95%, at least 97%, at least 99%, or100% sequence identity to SEQ ID NO: 104.

In some aspects, the programmable nuclease has at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 92%, at least95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:11 and the buffer comprises about 20 mM HEPES, about 2 mM potassiumacetate, about 5 mM magnesium acetate, about 1% glycerol, about 0.00016%Triton-X, and a pH of about 7.5. In some aspects, the programmablenuclease has at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 92%, at least 95%, at least 97%, at least 99%, or100% sequence identity to SEQ ID NO: 1 and the buffer comprises about 20mM Tris, about 100 mM sodium chloride, about 5 mM magnesium chloride,about 5% glycerol, about 50 ug/mL heparin, about 1 mM DTT, and a pH ofabout 8. In some aspects, the programmable nuclease has at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 95%, at least 97%, at least 99%, or 100% sequence identity to SEQID NO: 104 and the buffer comprises about 50 mM potassium chloride,about 5 mM magnesium chloride, about 10 ug/ml bovine serum albumin,about 5% (v/v) glycerol, about 20 mM imidazole, about 0.01% (v/v) IGEPALCA-630, and a pH of about 7.5. In some aspects, the composition furthercomprises a guide nucleic acid. In some aspects, the composition furthercomprises a detector nucleic acid.

In various aspects, the present disclosure provides a compositioncomprising: a nucleic acid from a sample, wherein the sample comprises aPAM and a segment that hybridizes to a guide nucleic acid, wherein thePAM has a sequence of dUdUdUN; a guide nucleic acid that hybridizes tothe segment of the nucleic acid; and a programmable nuclease thatexhibits sequence independent cleavage of a detector nucleic acid uponhybridization of the guide nucleic acid to the segment of the targetnucleic acid.

In some aspects, the composition further comprises a primer, wherein theprimer comprises a first region that is reverse complementary to the PAMand a second region that is reverse complementary to a first segment ofthe nucleic acid.

In various aspects, the present disclosure provides a method of assayingfor a target nucleic acid in a sample, wherein the target nucleic acidlacks a PAM, the method comprising: amplifying the target nucleic acidfrom a sample using a primer comprising a first region that is reversecomplementary to a PAM and a second region that is reverse complementaryto a first segment of the target nucleic acid, wherein the PAM isdUdUdUN, thereby producing a PAM target nucleic acid; contacting the PAMtarget nucleic acid to: a guide nucleic acid that hybridizes to asegment of the PAM target nucleic acid; a programmable nuclease thatexhibits sequence independent cleavage of a detector nucleic acid uponhybridization of the guide nucleic acid to a segment of the PAM targetnucleic acid; and a detector nucleic acid; and assaying for a signalproduced by cleavage of the detector nucleic acid.

In some aspects, the second region comprises from 4 to 12 bases. In someaspects, the second region comprises from 4 to 10 bases. In someaspects, the second region comprises from 4 to 7 bases. In some aspects,the amplifying comprises thermal cycling amplification. In some aspects,the amplifying comprises isothermal amplification. In some aspects, theisothermal amplification comprises isothermal recombinase polymeraseamplification (RPA), transcription mediated amplification (TMA), stranddisplacement amplification (SDA), helicase dependent amplification(HDA), loop mediated amplification (LAMP), rolling circle amplification(RCA), single primer isothermal amplification (SPIA), ligase chainreaction (LCR), simple method amplifying RNA targets (SMART), improvedmultiple displacement amplification (IMDA), or nucleic acidsequence-based amplification (NASBA). In some aspects, the isothermalamplification comprises loop mediated amplification (LAMP).

In some aspects, a sequence of the primer and a sequence of the guidenucleic acid overlap by 50% or less. In some aspects, a sequence of theprimer and a sequence of the guide nucleic acid do not overlap. In someaspects, the primer is a forward primer, a reverse primer, a forwardinner primer, or a reverse inner primer. In some aspects, the segment ofthe nucleic acid or the segment of the target nucleic acid comprises atleast one base mutation compared to at least one other segment of anucleic acid in the sample. In some aspects, the at least one basemutation is no more than 13 nucleotides 3′ of the PAM in the nucleicacid or the PAM target nucleic acid. In some aspects, the at least onebase mutation is no more than 10 nucleotides 3′ of the PAM in thenucleic acid or the PAM target nucleic acid. In some aspects, the atleast one base mutation is no more than 9 nucleotides 3′ of the PAM inthe nucleic acid or in the PAM target nucleic acid. In some aspects, theat least one base mutation is no more than 8 nucleotides 3′ of the PAMin the nucleic acid or in the PAM target nucleic acid. In some aspects,the at least one base mutation is a single nucleotide polymorphism.

In some aspects, the programmable nuclease comprises a RuvC domain. Insome aspects, the programmable nuclease comprises three partial RuvCdomains. In some aspects, wherein the programmable nuclease comprises aRuvC-I subdomain, a RuvC-II subdomain, and a RuvC-III subdomain. In someaspects, the programmable nuclease comprises a Type V Cas protein. Insome aspects, the programmable nuclease is a Cas12 protein. In someaspects, the Cas12 protein is Cas12a, Cas12b, Cas12c, CasY, or Cas12e.In some aspects, the programmable nuclease has at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 92%, at least95%, at least 97%, at least 99%, or 100% sequence identity to any one ofSEQ ID NO: 1-SEQ ID NO: 11, SEQ ID NO: 282, SEQ ID NO: 571-SEQ ID NO:602.

In various aspects, the present disclosure provides a Cas12 nuclease foruse in diagnosis, wherein the Cas12 nuclease detects the segment of thetarget nucleic acid according to any of the above methods.

In some aspects, the present disclosure provides for the use of any ofthe above compositions in diagnosis.

In various aspects, the present disclosure provides for a programmablenuclease for use in diagnosis, wherein the programmable nuclease detectsthe target nucleic acid according to any of the above described methods.

Provided herein are embodiments related to improved Cas12, Cas13, andCas14 proteins and related compositions and methods of use. Embodimentsare summarized in part in the claims as listed herein.

In various aspects, the present disclosure provides a programmablenuclease that elicits maximal reporter activity no more than 60 minutesfollowing contacting to a target template at a target templateconcentration of 100 nM.

In some aspects, the programmable nuclease comprises a Cas12 protein, aCas13 protein, or a Cas14 protein. In some aspects, said protein elicitsmaximal reporter activity following contacting to a target template atleast 50% faster than LbCas12a at a given target template concentration.In some aspects, said protein elicits maximal reporter activityfollowing contacting to a target template at least 2× faster thanLbCas12a at a given target template concentration. In some aspects, saidprotein elicits maximal reporter activity following contacting to atarget template at least 4× faster than LbCas12a at a given targettemplate concentration. In some aspects, said protein elicits no greaterthan 33% of maximal reporter activity following contacting to a templatediffering from a target template by a single base at a templateconcentration of 100 nM. In some aspects, the protein elicits maximalreporter activity in a composition comprising at least one componentselected from the list consisting of acetate, heparin, dithiothreitol(DTT), triton-X, TCEP, BSA, NP-40, imidazole, MOPS, HEPES and DIPSO.

In some aspects, the template is unamplified. In some aspects, thetemplate is amplified prior to contacting. In some aspects, thecontacting is performed in an activity buffer (5×: 600 mM NaCl, 25 mMMgCl2, 100 mM Tris pH 7.5, 5% (v/v) glycerol). In some aspects, thecontacting is performed at about 25° C. In some aspects, the contactingis performed at about 37° C.

In various aspects, the present disclosure provides a programmablenuclease reaction buffer comprising at least one component selected fromthe list consisting of acetate, heparin, dithiothreitol (DTT), triton-X,TCEP, BSA, NP-40, imidazole, MOPS, HEPES and DIPSO.

In some aspects, the programmable nuclease comprises a Cas12 protein, aCas13 protein, or a Cas14 protein. In some aspects, the programmablenuclease in said reaction buffer elicits no greater than 33% of maximalreporter activity following contacting to a template differing from atarget template by a single base. In some aspects, the reaction buffercomprises no greater than 150 mM NaCl. In some aspects, the reactionbuffer comprises no greater than 100 mM NaCl. In some aspects, thereaction buffer comprises no greater than 50 mM NaCl. In some aspects,the reaction buffer comprises no greater than 25 mM NaCl.

In various aspects, the present disclosure provides a programmablenuclease reaction buffer comprising at least one component selected fromthe list consisting of DMSO, polyvinyl alcohol, polyvinylpyrrolidone,and polypropylene glycol.

In some aspects, the programmable nuclease comprises a Cas12 protein, aCas13 protein, or a Cas14 protein. In some aspects, the programmablenuclease in said reaction buffer elicits no greater than 33% of maximalreporter activity following contacting to a no-template control. In someaspects, the reaction buffer comprises no greater than 150 mM NaCl. Insome aspects, the reaction buffer comprises no greater than 100 mM NaCl.In some aspects, the reaction buffer comprises no greater than 50 mMNaCl. In some aspects, the reaction buffer comprises no greater than 25mM NaCl.

In various aspects, the present disclosure provides a programmablenuclease that elicits reporter activity no more than 60 minutesfollowing contacting to a target template at a target templateconcentration of 1 nM in an activity buffer (5×: 600 mM NaCl, 25 mMMgCl2, 100 mM Tris pH 7.5, 5% (v/v) glycerol).

In some aspects, the programmable nuclease comprises a Cas12 protein, aCas13 protein, or a Cas14 protein. In some aspects, the Cas12 proteinelicits reporter activity no more than 60 minutes following contactingto a target template at a target template concentration of 1 pM. In someaspects, the Cas12 protein elicits reporter activity no more than 60minutes following contacting to a target template at a target templateconcentration of 1 fM.

In various aspects, the present disclosure provides a programmablenuclease that exhibits at least 90% target cleavage in no more than 60minutes.

In some aspects, the programmable nuclease comprises a Cas12 protein, aCas13 protein, or a Cas14 protein. In some aspects, the Cas12 proteinexhibits at least 90% target cleavage in no more than 15 minutes. Insome aspects, an activity buffer (5×: 600 mM NaCl, 25 mM MgCl2, 100 mMTris pH 7.5, 5% (v/v) glycerol) exhibits said target cleavage. In someaspects, said target cleavage is effected at a Cas12 concentration of100 nM. In some aspects, said target cleavage is effected at a targetconcentration of 15 nM. In some aspects, said target cleavage iseffected at a guide RNA concentration of 125 nM. In some aspects, saidtarget cleavage is effected at a temperature of about 25° C. In someaspects, said target cleavage is effected at a temperature of about 37°C.

In various aspects, the present disclosure provides a programmablenuclease that exhibits no more than 10% target cleavage in 60 minutes.

In some aspects, the programmable nuclease comprises a Cas12 protein, aCas13 protein, or a Cas14 protein. In some aspects, an activity buffer(5×: 600 mM NaCl, 25 mM MgCl2, 100 mM Tris pH 7.5, 5% (v/v) glycerol)exhibits said target cleavage. In some aspects, said target cleavage iseffected at a Cas12 concentration of 100 nM. In some aspects, saidtarget cleavage is effected at a target concentration of 15 nM. In someaspects, said target cleavage is effected at a guide RNA concentrationof 125 nM. In some aspects, said target cleavage is effected at atemperature of about 25° C. In some aspects, said target cleavage iseffected at a temperature of about 37° C.

In various aspects, the present disclosure provides a compositioncomprising a first programmable nuclease population and a secondprogrammable nuclease population, wherein the first programmablenuclease population and the second programmable nuclease population donot share a common PAM sequence.

In some aspects, the composition comprises a third programmable nucleasepopulation, wherein none of the first programmable nuclease population,the second programmable nuclease population, and the third programmablenuclease population share a common PAM sequence. In some aspects, thecomposition comprises a fourth programmable nuclease population, whereinnone of the first programmable nuclease population, the secondprogrammable nuclease population, the third programmable nucleasepopulation, and the programmable nuclease Cas12 population share acommon PAM sequence. In some aspects, the first programmable nuclease,the second programmable nuclease, or a combination thereof comprises aCas12 protein, a Cas13 protein, or a Cas14 protein. In some aspects, thethird programmable nuclease comprises a Cas12 protein, a Cas13 protein,or a Cas14 protein. In some aspects, the fourth programmable nucleasecomprises a Cas12 protein, a Cas13 protein, or a Cas14 protein.

In various aspects, the present disclosure provides a method forcleaving a unique site of a nucleic acid molecule, comprising designinga guide nucleic acid to cleave the unique site of the nucleic acidmolecule and contacting the guide nucleic acid to a programmablenuclease and to the unique site of the nucleic acid molecule, therebycleaving the unique site of the nucleic acid molecule.

In some aspects, a PAM sequence is not considered in the designing ofthe guide nucleic acid. In some aspects, the programmable nucleasecomprises a Cas protein. In some aspects, the Cas protein is Cas14.

In various aspects, the present disclosure provides a method of sequencespecific cleavage of a nucleic acid molecule in a sample comprisingcontacting to a first PAM independent nuclease to a flank on one side ofa cleavage site the nucleic acid molecule and a second PAM independentnuclease to a flank on the other side of the cleavage site of thenucleic acid molecule.

In some aspects, the method further comprises contacting the sample to aDNA fragment for sequence specific break repair. In some aspects, thePAM independent nuclease is a Cas protein. In some aspects, the Casprotein is a nickase. In some aspects, the Cas protein is Cas14.

In various aspects, the present disclosure provides a method ofdetecting a presence or an absence of a target nucleic acid in a sample,the method comprising: contacting a first volume to a second volume,wherein the first volume comprises the sample and the second volumecomprises: i) a guide nucleic acid having at least 10 nucleotidesreverse complementary to a target nucleic acid in the sample; and ii) aprogrammable nuclease activated upon binding of the guide nucleic acidto the target nucleic acid; iii) a reporter comprising a nucleic acidand a detection moiety, wherein the second volume is at least 4-foldgreater than the first volume; and detecting the presence or the absenceof the target nucleic acid by measuring a signal produced by cleavage ofthe nucleic acid of the reporter, wherein cleavage occurs when theprogrammable nuclease is activated.

In some aspects, the first volume comprises from 1 μL to 10 μL. In someaspects, the first volume comprises from 1 μL to 5 μL. In some aspects,the first volume comprises about 2 μL. In some aspects, the first volumecomprises about 4 μL. In some aspects, the second volume comprises from5 μL to 40 μL. In some aspects, the second volume comprises from 10 μLto 30 μL. In some aspects, the second volume comprises about 20 μL. Insome aspects, the second volume comprises about 30 μL.

In some aspects, the sample first volume comprises a buffer for celllysis, a buffer for amplification, a primer, a polymerase, targetnucleic acid, a non-target nucleic acid, a single-stranded DNA, adouble-stranded DNA, a salt, a buffering agent, an NTP, a dNTP, or anycombination thereof. In some aspects, the sample is a biological samplecomprising blood, serum, plasma, saliva, urine, mucosal sample,peritoneal sample, cerebrospinal fluid, gastric secretions, nasalsecretions, sputum, pharyngeal exudates, urethral or vaginal secretions,an exudate, an effusion, or tissue.

In some aspects, the programmable nuclease is a programmable Type VCRISPR/Cas enzyme. In some aspects, the programmable Type V CRISPR/Casenzyme is a programmable Cas12 nuclease. In some aspects, theprogrammable Cas12 nuclease is Cas12a, Cas12b, Cas12c, Cas12d, orCas12e. In some aspects, the programmable Type V CRISPR/Cas enzyme is aprogrammable Cas14 nuclease. In some aspects, the programmable Cas14nuclease is Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, orCas14h. In some aspects, the programmable nuclease is a programmableType VI CRISPR/Cas enzyme. In some aspects, the programmable Type VICRISPR/Cas enzyme is a programmable Cas13 nuclease. In some aspects, theprogrammable Cas13 nuclease is Cas13a, Cas13b, Cas13c, Cas13d, orCas13e.

In various aspects, the present disclosure provides a method ofdesigning a plurality of primers for amplification of a target nucleicacid, the method comprising: providing a target nucleic acid, herein aguide nucleic acid hybridizes to the target nucleic acid and wherein atleast 60% of a sequence of the target nucleic acid is between an F1cregion and a B1 region or between an F1 and a B1c region; and designingthe plurality of primers comprising: i) a forward inner primercomprising a sequence of the F1c region 5′ of a sequence of an F2region; ii) a backward inner primer comprising a sequence of the B1cregion 5′ of a sequence of a B2 region; iii) a forward outer primercomprising a sequence of an F3 region; and iv) a backward outer primercomprising a sequence of a B3 region.

In various aspects, the present disclosure provides a method ofdetecting a target nucleic acid in a sample, the method comprising:contacting the sample to: a plurality of primers comprising: i) aforward inner primer comprising a sequence corresponding to an F1cregion 5′ of a sequence corresponding to an F2 region; ii) a backwardinner primer comprising a sequence corresponding to a B1c region 5′ of asequence corresponding to a B2 region; iii) a forward outer primercomprising a sequence corresponding to an F3 region; and iv) a backwardouter primer comprising a sequence corresponding to a B3 region; a guidenucleic acid, wherein the guide nucleic acid hybridizes to the targetnucleic acid and wherein at least 60% of a sequence of the targetnucleic acid is between the F1c region and a B1 region or between an F1region and the B1c region; a reporter; and a programmable nuclease thatcleaves the reporter when complexed with the guide nucleic acid; and

measuring a detectable signal produced by cleavage of the reporter,wherein the measuring provides for detection of the target nucleic acidin the sample.

In some aspects, the sequence between the F1c region and the B1 regionor the sequence between the B1c region and the F1 region is at least 50%reverse complementary to the guide nucleic acid sequence. In someaspects, the guide nucleic acid sequence is reverse complementary to nomore than 50% of the forward inner primer, the backward inner primer, ora combination thereof. In some aspects, the guide nucleic acid does nothybridize to the forward inner primer and the backward inner primer.

In some aspects, a protospacer adjacent motif (PAM) or a protospacerflanking site (PFS) is 3′ of the target nucleic acid. In some aspects, aprotospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is3′ of the B1 region and 5′ of the F1c region or the protospacer adjacentmotif (PAM) or a protospacer flanking site (PFS) is 3′ of the F1 regionand 5′ of the B1c region. In some aspects, the 3′ end of the targetnucleic acid is 5′ of the 5′ end of the F3c region or the 3′ end of thetarget nucleic acid is 5′ of the 5′ end of the B3c region. In someaspects, the 3′ end of the target nucleic acid is 5′ of the 5′ end ofthe F2c region or 3′ end of the target nucleic acid is 5′ of the 5′ endof the B2c region. In some aspects, the target nucleic acid is betweenthe F1c region and the B1 region and the 3′ end of the target nucleicacid is 5′ of the 3′ end of the F2c region, or wherein the targetnucleic acid is between the B1c region and the F1 region and the 3′ endof the target nucleic acid is 5′ of the 3′ end of the B2c region.

In some aspects, the guide nucleic acid has a sequence reversecomplementary to no more than 50% of the forward inner primer, thebackward inner primer, the forward outer primer, the backward outerprimer, or any combination thereof. In some aspects, the guide nucleicacid sequence does not hybridize to the forward inner primer, thebackward inner primer, the forward outer primer, the backward outerprimer, or any combination thereof.

In some aspects, the guide nucleic acid sequence has a sequence reversecomplementary to no more than 50% of a sequence of an F3c region, an F2cregion, the F1c region, the B1c region, an B2c region, an B3c region, orany combination thereof. In some aspects, the guide nucleic acidsequence does not hybridize to a sequence of an F3c region, an F2cregion, the F1c region, the B1c region, an B2c region, an B3c region, orany combination thereof.

In various aspects, the present disclosure provides a method ofdesigning a plurality of primer for amplification of a target nucleicacid, the method comprising: providing the target nucleic acidcomprising a sequence between a B2 region and a B1 region or between anF2 region and an F1 region that hybridizes to a guide nucleic acid; anddesigning the plurality of primers comprising: i) a forward inner primercomprising a sequence of the F1c region 5′ of a sequence of an F2region; ii) a backward inner primer comprising a sequence of the B1cregion 5′ of a sequence of a B2 region; iii) a forward outer primercomprising a sequence of an F3 region; and iv) a backward outer primercomprising a sequence of a B3 region.

In various aspects, the present disclosure provides a method ofdesigning a plurality of primer for amplification of a target nucleicacid, the method comprising: providing the target nucleic acidcomprising a sequence between a F1c region and an F2c region or betweena B1c region and a B2c region that hybridizes to a guide nucleic acid;and designing the plurality of primers comprising: i) a forward innerprimer comprising a sequence of the F1c region 5′ of a sequence of an F2region; ii) a backward inner primer comprising a sequence of the B1cregion 5′ of a sequence of a B2 region; iii) a forward outer primercomprising a sequence of an F3 region; and iv) a backward outer primercomprising a sequence of a B3 region.

In various aspects, the present disclosure provides a method ofdetecting a target nucleic acid in a sample, the method comprising:contacting the sample to: a plurality of primers comprising: i) aforward inner primer comprising a sequence corresponding to an F1cregion 5′ of a sequence corresponding to an F2 region; ii) a backwardinner primer comprising a sequence corresponding to a B1c region 5′ of asequence corresponding to a B2 region; iii) a forward outer primercomprising a sequence corresponding to an F3 region; and iv) a backwardouter primer comprising a sequence corresponding to a B3 region; a guidenucleic acid, wherein the target nucleic acid comprises a sequencebetween a B2 region and a B1 region or between the F2 region and an F1region that hybridizes to the guide nucleic acid; a reporter; and aprogrammable nuclease that cleaves the reporter when complexed with theguide nucleic acid; and measuring a detectable signal produced bycleavage of the reporter, wherein the measuring provides for detectionof the target nucleic acid in the sample.

In various aspects, the present disclosure provides a method ofdetecting a target nucleic acid in a sample, the method comprising:contacting the sample to: a plurality of primers comprising: i) aforward inner primer comprising a sequence corresponding to an F1cregion 5′ of a sequence corresponding to an F2 region; ii) a backwardinner primer comprising a sequence corresponding to a B1c region 5′ of asequence corresponding to a B2 region; iii) a forward outer primercomprising a sequence corresponding to an F3 region; and iv) a backwardouter primer comprising a sequence corresponding to a B3 region; a guidenucleic acid, wherein the target nucleic acid comprises a sequencebetween the F1c region and an F2c region or between the B1c region and aB2c region that hybridizes to the guide nucleic acid; a reporter; and aprogrammable nuclease that cleaves the reporter when complexed with theguide nucleic acid; and measuring a detectable signal produced bycleavage of the reporter, wherein the measuring provides for detectionof the target nucleic acid in the sample.

In some aspects, a protospacer adjacent motif (PAM) or a protospacerflanking site (PFS) is 3′ of the B2 region and 5′ of the B1 region orthe protospacer adjacent motif (PAM) or a protospacer flanking site(PFS) is 3′ of the F2 region and 5′ of the F1 region. In some aspects, aprotospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is3′ of the B1c region and 5′ of the B2c region or the protospaceradjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of theF1c region and 5′ of the F2c region.

In some aspects, a protospacer adjacent motif (PAM) or a protospacerflanking site (PFS) is 3′ of the target nucleic acid. In some aspects,the PAM and the PFS are 5′ of the 5′ end of the F1c region, 5′ of the 5′end of the B1c region, 3′ of the 3′ end of the F3 region, 3′ of the 3′end of the B3 region, 3′ of the 3′ end of the F2 region, 3′ of the 3′end of the B2 region, or any combination thereof.

In some aspects, the PAM and the PFS do not overlap the F2 region, theB3 region, the F1c region, the F2 region, the B1c region, the B2 region,or any combination thereof. In some aspects, the PAM and the PFS do nothybridize to the forward inner primer, the backward inner primer, theforward outer primer, the backward outer primer, or any combinationthereof.

In some aspects, the plurality of primers further comprises a loopforward primer. In some aspects, the plurality of primers furthercomprises a loop backward primer. In some aspects, the loop forwardprimer is between an F1c region and an F2c region. In some aspects, theloop backward primer is between a B1c region and a B2c region.

In some aspects, the target nucleic acid comprises a single nucleotidepolymorphism (SNP). In some aspects, the single nucleotide polymorphism(SNP) comprises a HERC2 SNP. In some aspects, the single nucleotidepolymorphism (SNP) is associated with an increased risk or decreasedrisk of cancer. In some aspects, the target nucleic acid comprises asingle nucleotide polymorphism (SNP), and wherein the detectable signalis higher in the presence of a guide nucleic acid that is 100%complementary to the target nucleic acid comprising the singlenucleotide polymorphism (SNP) than in the presence of a guide nucleicacid that is less than 100% complementary to the target nucleic acidcomprising the single nucleotide polymorphism (SNP).

In some aspects, the plurality of primers and the guide nucleic acid arepresent together in a sample comprising the target nucleic acid. In someaspects, the contacting the sample to the plurality of primers resultsin amplifying the target nucleic acid. In some aspects, the amplifyingand the contacting the sample to the guide nucleic acid occurs at thesame time. In other aspects, the amplifying and the contacting thesample to the guide nucleic acid occur at different times. In someaspects, the method further comprises providing a polymerase, a dATP, adTTP, a dGTP, a dCTP, or any combination thereof.

The present disclosure provides methods of detecting a target nucleicacid using a programmable nuclease.

In some aspects, the present disclosure provides a method of assayingfor a target nucleic acid in a sample, comprising: contacting the sampleto a complex comprising a guide nucleic acid comprising a segment thatis reverse complementary to a segment of the target nucleic acid and aprogrammable nuclease that exhibits sequence independent cleavage uponforming a complex comprising the segment of the guide nucleic acidbinding to the segment of the target nucleic acid, wherein the samplecomprises at least one nucleic acid comprising at least 50% sequenceidentity to the segment of the target nucleic acid; and assaying forcleavage of at least one detector nucleic acids of a population ofdetector nucleic acids, wherein the cleavage indicates a presence of thetarget nucleic acid in the sample and wherein absence of the cleavageindicates an absence of the target nucleic acid in the sample. Often,the target nucleic acid is from 0.05% to 20% of total nucleic acids inthe sample. Various strategies, such as amplifying the target nucleic toinsert a PAM sequence, COLD-PCR, allele-specific PCR, targeting thenucleic acid with a protein, or targeting other nucleic acids withprotein can be used to enrich for the target nucleic acid in the sample.Additionally, a buffer comprising NaCl and heparin enhances thespecificity of the programmable nuclease in the methods provided herein.

In various aspects, the present disclosure provides a method of assayingfor a target nucleic acid in a sample, comprising: contacting the sampleto a complex comprising a guide nucleic acid comprising a segment thatis reverse complementary to a segment of the target nucleic acid and aprogrammable nuclease that exhibits sequence independent cleavage uponforming a complex comprising the segment of the guide nucleic acidbinding to the segment of the target nucleic acid, wherein the samplecomprises at least one nucleic acid comprising at least 50% sequenceidentity to the segment of the target nucleic acid; and assaying forcleavage of at least one detector nucleic acids of a population ofdetector nucleic acids, wherein the cleavage indicates a presence of thetarget nucleic acid in the sample and wherein absence of the cleavageindicates an absence of the target nucleic acid in the sample.

In some aspects, the target nucleic acid is from 0.05% to 20% of totalnucleic acids in the sample. In further aspects, the target nucleic acidis from 0.1% to 10% of total nucleic acids in the sample. In stillfurther aspects, the target nucleic acid is from 0.1% to 5% of totalnucleic acids in the sample. In some aspects, the contacting isperformed in a buffer comprising heparin and NaCl. In further aspects,the NaCl is 100 mM NaCl. In some aspects, the heparin is 50 ug/mlheparin.

In some aspects, the sample comprises at least one nucleic acidcomprising at least 80% sequence identity to the segment of the targetnucleic acid. In further aspects, the sample comprises at least onenucleic acid comprising at least 90% sequence identity to the segment ofthe target nucleic acid. In still further aspects, the sample comprisesat least one nucleic acid comprising at least 99% sequence identity tothe segment of the target nucleic acid.

In some aspects, the sample comprises at least one nucleic acidcomprising less than 100% sequence identity to the segment of the targetnucleic acid and no less than 50% sequence identity to the segment ofthe target nucleic acid. In some aspects, the sample comprises at leastone nucleic acid comprising less than 100% sequence identity to thesegment of the target nucleic acid and no less than 80% sequenceidentity to the segment of the target nucleic acid. In some aspects, thesample comprises at least one nucleic acid comprising less than 100%sequence identity to the segment of the target nucleic acid and no lessthan 90% sequence identity to the segment of the target nucleic acid.

In some aspects, the target nucleic acid comprises a single nucleotidemutation. In further aspects, the segment of the target nucleic acidcomprises a single nucleotide mutation. In some aspects, the singlenucleotide mutation is a synonymous substitution or a nonsynonymoussubstitution. In some aspects, the nonsynonymous substitution is amissense substitution or a nonsense point mutation.

In other aspects, the target nucleic acid comprises a deletion. Infurther aspects, the segment of the target nucleic acid comprises adeletion. In some aspects, the deletion comprises a deletion of from 1to 50 nucleotides. In some aspects, the deletion comprises a deletion offrom 9 to 21 nucleotides.

In some aspects, the method further comprises amplifying the targetnucleic acid segment using a primer having a region that is reversecomplementary to the target nucleic acid segment and a region that has aPAM sequence reverse complement, thereby generating a PAM target nucleicacid having a PAM sequence adjacent to target sequence of anamplification product before the contacting. In some aspects, the primeris a forward primer comprising the sequence encoding the PAM and has 1-8nucleotides from the 3′ end of the sequence encoding the PAM. In someaspects, the primer is a forward primer comprising the sequence encodingthe PAM and has 4 nucleotides from the 3′ end of the sequence encodingthe PAM.

In other aspects, the primer is a forward primer comprising the sequenceencoding the PAM and has 5 nucleotides from the 3′ end of the sequenceencoding the PAM. In still other aspects, the primer is a forward primercomprising the sequence encoding the PAM and has 6 nucleotides from the3′ end of the sequence encoding the PAM.

In some aspects, the segment of the target nucleic acid comprises thesingle nucleotide mutation at 5-9 nucleotides downstream of the 5′ endthe segment of the target nucleic acid comprising the sequence theencoding the PAM. In some aspects, the segment of the target nucleicacid comprises the single nucleotide mutation at 6 nucleotidesdownstream of the 5′ end the segment of the target nucleic acidcomprising the sequence the encoding the PAM. In other aspects, thesegment of the target nucleic acid comprises the single nucleotidemutation at 7 nucleotides downstream of the 5′ end the segment of thetarget nucleic acid comprising the sequence the encoding the PAM. Instill other aspects, the segment of the target nucleic acid comprisesthe single nucleotide mutation at 8 nucleotides downstream of the 5′ endthe segment of the target nucleic acid comprising the sequence theencoding the PAM.

In some aspects, the segment of the target nucleic acid comprises thedeletion at 5-9 nucleotides downstream of the 5′ end the segment of thetarget nucleic acid comprising the sequence the encoding the PAM. Insome aspects, the segment of the target nucleic acid comprises thedeletion at 6 nucleotides downstream of the 5′ end the segment of thetarget nucleic acid comprising the sequence the encoding the PAM. Inother aspects, the segment of the target nucleic acid comprises thedeletion at 7 nucleotides downstream of the 5′ end the segment of thetarget nucleic acid comprising the sequence the encoding the PAM. Instill other aspects, the segment of the target nucleic acid comprisesthe deletion at 8 nucleotides downstream of the 5′ end the segment ofthe target nucleic acid comprising the sequence the encoding the PAM.

In some aspects, the method further comprises amplifying the targetnucleic acid before the contacting. In some aspects, the amplifying thetarget nucleic acid before the contacting comprises using a blockingprimer. In some aspects, the blocking primer binds to a nucleic acidcomprising encoding the wild type sequence of the target nucleic acidsegment. In some aspects, the amplifying comprises COLD-PCR.

In further aspects, the COLD-PCR comprises full COLD-PCR. In someaspects, the COLD-PCR comprises fast COLD-PCR. In some aspects, theamplifying comprises fast COLD-PCR. In some aspects, the amplifyingcomprises allele-specific PCR. In some aspects, the amplifying furthercomprises COLD-PCR.

In some aspects, the method further comprises removing a nucleic acidcomprising at least 50% sequence identity to the target nucleic acid bybinding a protein to the nucleic acid before the contacting. In someaspects, the protein is an antibody. In some aspects, the protein is aprogrammable nuclease without endonuclease activity. In some aspects,the method further comprises binding a protein to the target nucleicacid to remove other nucleic acids of the sample. In some aspects, theother nucleic acids comprise a nucleic acid comprising at least 50%sequence identity to the target nucleic acid. In some aspects, theprotein is attached to a surface. In some aspects, the removing of theother nucleic acids comprises washing away nucleic acids that are notbound to the protein. In some aspects, the protein is an antibody. Insome aspects, the protein is a programmable nuclease withoutendonuclease activity.

In some aspects, the programmable nuclease is a target nucleic acidactivated effector protein that exhibits sequence independent cleavageupon activation. In some aspects, the programmable nuclease is an RNAguided nuclease. In some aspects, the programmable nuclease comprises aCas nuclease. In some aspects, the Cas nuclease is Cas13. In furtheraspects, the Cas13 is Cas13a, Cas13b, Cas13c, Cas13d, or Cas13e. Inother aspects, the Cas nuclease is Cas12. In further aspects, the Cas12is Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e. In some aspects, the Casnuclease is Cas14. In further aspects, the Cas14 is Cas14a, Cas14b,Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, or Cas14h. In some aspects, theCas nuclease is Csm1, Cas9, C2c4, C2c8, C2c5, C2c10, or C2c9.

In some aspects, the guide nucleic acid comprises a crRNA. In someaspects, the guide nucleic acid comprises a crRNA and a tracrRNA. Insome aspects, cleavage of at least one detector nucleic acid yields asignal. In some aspects, cleavage of at least one detector nucleic acidactivates a photoexcitable fluorophore. In some aspects, cleavage of atleast one detector nucleic acid deactivates a photoexcitablefluorophore. In some aspects, the signal is present prior to detectornucleic acid cleavage. In some aspects, the signal is absent prior todetector nucleic acid cleavage.

In some aspects, the sample comprises blood, serum, plasma, saliva,urine, mucosal sample, peritoneal sample, cerebrospinal fluid, gastricsecretions, nasal secretions, sputum, pharyngeal exudates, urethral orvaginal secretions, an exudate, an effusion, or tissue. In some aspects,the single nucleotide mutation is a single nucleotide polymorphism.

In various aspects, the present disclosure provides a method,comprising: contacting a programmable nuclease comprising a polypeptidehaving endonuclease activity and a guide nucleic acid to a targetnucleic acid in a buffer comprising heparin. In some aspects, theheparin is present at a concentration of from 1 to 100 ug/ml heparin. Infurther aspects, the heparin is present at a concentration of from 40 to60 ug/ml heparin. In still further aspects, the heparin is present at aconcentration 50 ug/ml heparin.

In some aspects, the buffer comprises NaCl. In further aspects, the NaClis present at a concentration of from 1 to 200 mM NaCl. In still furtheraspects, the NaCl is present at a concentration of from 80 to 120 mMNaCl. In still further aspects, the NaCl is present at a concentrationof 100 mM NaCl.

In some aspects, the target nucleic acid is a substrate target nucleicacid. In some aspects, the substrate nucleic acid comprises a cancerallele. In further aspects, the cancer allele is present at a lowconcentration relative to a wild type allele. In some aspects, thesubstrate target nucleic acid comprises a splice variant. In someaspects, the substrate target nucleic acid comprises an edited base. Insome aspects, the substrate target nucleic acid comprises abisulfite-treated base. In some aspects, the substrate target nucleicacid comprises a segment that is reverse complementary to a segment ofthe guide nucleic acid.

In various aspects, the present disclosure provides a method ofdesigning a plurality of primers for amplification of a target nucleicacid, the method comprising: providing a target nucleic acid, herein aguide nucleic acid hybridizes to the target nucleic acid and wherein atleast 60% of a sequence of the target nucleic acid is between an F1cregion and a B1 region or between an F1 and a B1c region; and designingthe plurality of primers comprising: i) a forward inner primercomprising a sequence of the F1c region 5′ of a sequence of an F2region; ii) a backward inner primer comprising a sequence of the B1cregion 5′ of a sequence of a B2 region; iii) a forward outer primercomprising a sequence of an F3 region; and iv) a backward outer primercomprising a sequence of a B3 region.

In various aspects, the present disclosure provides a method ofdetecting a target nucleic acid in a sample, the method comprising:contacting the sample to: a plurality of primers comprising: i) aforward inner primer comprising a sequence corresponding to an F1cregion 5′ of a sequence corresponding to an F2 region; ii) a backwardinner primer comprising a sequence corresponding to a B1c region 5′ of asequence corresponding to a B2 region; iii) a forward outer primercomprising a sequence corresponding to an F3 region; and iv) a backwardouter primer comprising a sequence corresponding to a B3 region; a guidenucleic acid, wherein the guide nucleic acid hybridizes to the targetnucleic acid and wherein at least 60% of a sequence of the targetnucleic acid is between the F1c region and a B1 region or between an F1region and the B1c region; a reporter; and a programmable nuclease thatcleaves the reporter when complexed with the guide nucleic acid; and

measuring a detectable signal produced by cleavage of the reporter,wherein the measuring provides for detection of the target nucleic acidin the sample.

In some aspects, the sequence between the F1c region and the B1 regionor the sequence between the B1c region and the F1 region is at least 50%reverse complementary to the guide nucleic acid sequence. In someaspects, the guide nucleic acid sequence is reverse complementary to nomore than 50% of the forward inner primer, the backward inner primer, ora combination thereof. In some aspects, the guide nucleic acid does nothybridize to the forward inner primer and the backward inner primer.

In some aspects, a protospacer adjacent motif (PAM) or a protospacerflanking site (PFS) is 3′ of the target nucleic acid. In some aspects, aprotospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is3′ of the B1 region and 5′ of the F1c region or the protospacer adjacentmotif (PAM) or a protospacer flanking site (PFS) is 3′ of the F1 regionand 5′ of the B1c region. In some aspects, the 3′ end of the targetnucleic acid is 5′ of the 5′ end of the F3c region or the 3′ end of thetarget nucleic acid is 5′ of the 5′ end of the B3c region. In someaspects, the 3′ end of the target nucleic acid is 5′ of the 5′ end ofthe F2c region or 3′ end of the target nucleic acid is 5′ of the 5′ endof the B2c region. In some aspects, the target nucleic acid is betweenthe F1c region and the B1 region and the 3′ end of the target nucleicacid is 5′ of the 3′ end of the F2c region, or wherein the targetnucleic acid is between the B1c region and the F1 region and the 3′ endof the target nucleic acid is 5′ of the 3′ end of the B2c region.

In some aspects, the guide nucleic acid has a sequence reversecomplementary to no more than 50% of the forward inner primer, thebackward inner primer, the forward outer primer, the backward outerprimer, or any combination thereof. In some aspects, the guide nucleicacid sequence does not hybridize to the forward inner primer, thebackward inner primer, the forward outer primer, the backward outerprimer, or any combination thereof.

In some aspects, the guide nucleic acid sequence has a sequence reversecomplementary to no more than 50% of a sequence of an F3c region, an F2cregion, the F1c region, the B1c region, an B2c region, an B3c region, orany combination thereof. In some aspects, the guide nucleic acidsequence does not hybridize to a sequence of an F3c region, an F2cregion, the F1c region, the B1c region, an B2c region, an B3c region, orany combination thereof.

In various aspects, the present disclosure provides a method ofdesigning a plurality of primer for amplification of a target nucleicacid, the method comprising: providing the target nucleic acidcomprising a sequence between a B2 region and a B1 region or between anF2 region and an F1 region that hybridizes to a guide nucleic acid; anddesigning the plurality of primers comprising: i) a forward inner primercomprising a sequence of the F1c region 5′ of a sequence of an F2region; ii) a backward inner primer comprising a sequence of the B1cregion 5′ of a sequence of a B2 region; iii) a forward outer primercomprising a sequence of an F3 region; and iv) a backward outer primercomprising a sequence of a B3 region.

In various aspects, the present disclosure provides a method ofdesigning a plurality of primer for amplification of a target nucleicacid, the method comprising: providing the target nucleic acidcomprising a sequence between a F1c region and an F2c region or betweena B1c region and a B2c region that hybridizes to a guide nucleic acid;and designing the plurality of primers comprising: i) a forward innerprimer comprising a sequence of the F1c region 5′ of a sequence of an F2region; ii) a backward inner primer comprising a sequence of the B1cregion 5′ of a sequence of a B2 region; iii) a forward outer primercomprising a sequence of an F3 region; and iv) a backward outer primercomprising a sequence of a B3 region.

In various aspects, the present disclosure provides a method ofdetecting a target nucleic acid in a sample, the method comprising:contacting the sample to: a plurality of primers comprising: i) aforward inner primer comprising a sequence corresponding to an F1cregion 5′ of a sequence corresponding to an F2 region; ii) a backwardinner primer comprising a sequence corresponding to a B1c region 5′ of asequence corresponding to a B2 region; iii) a forward outer primercomprising a sequence corresponding to an F3 region; and iv) a backwardouter primer comprising a sequence corresponding to a B3 region; a guidenucleic acid, wherein the target nucleic acid comprises a sequencebetween a B2 region and a B1 region or between the F2 region and an F1region that hybridizes to the guide nucleic acid; a reporter; and aprogrammable nuclease that cleaves the reporter when complexed with theguide nucleic acid; and measuring a detectable signal produced bycleavage of the reporter, wherein the measuring provides for detectionof the target nucleic acid in the sample.

In various aspects, the present disclosure provides a method ofdetecting a target nucleic acid in a sample, the method comprising:contacting the sample to: a plurality of primers comprising: i) aforward inner primer comprising a sequence corresponding to an F1cregion 5′ of a sequence corresponding to an F2 region; ii) a backwardinner primer comprising a sequence corresponding to a B1c region 5′ of asequence corresponding to a B2 region; iii) a forward outer primercomprising a sequence corresponding to an F3 region; and iv) a backwardouter primer comprising a sequence corresponding to a B3 region; a guidenucleic acid, wherein the target nucleic acid comprises a sequencebetween the F1c region and an F2c region or between the B1c region and aB2c region that hybridizes to the guide nucleic acid; a reporter; and aprogrammable nuclease that cleaves the reporter when complexed with theguide nucleic acid; and measuring a detectable signal produced bycleavage of the reporter, wherein the measuring provides for detectionof the target nucleic acid in the sample.

In some aspects, a protospacer adjacent motif (PAM) or a protospacerflanking site (PFS) is 3′ of the B2 region and 5′ of the B1 region orthe protospacer adjacent motif (PAM) or a protospacer flanking site(PFS) is 3′ of the F2 region and 5′ of the F1 region. In some aspects, aprotospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is3′ of the B1c region and 5′ of the B2c region or the protospaceradjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of theF1c region and 5′ of the F2c region.

In some aspects, a protospacer adjacent motif (PAM) or a protospacerflanking site (PFS) is 3′ of the target nucleic acid. In some aspects,the PAM and the PFS are 5′ of the 5′ end of the F1c region, 5′ of the 5′end of the B1c region, 3′ of the 3′ end of the F3 region, 3′ of the 3′end of the B3 region, 3′ of the 3′ end of the F2 region, 3′ of the 3′end of the B2 region, or any combination thereof.

In some aspects, the PAM and the PFS do not overlap the F2 region, theB3 region, the F1c region, the F2 region, the B1c region, the B2 region,or any combination thereof. In some aspects, the PAM and the PFS do nothybridize to the forward inner primer, the backward inner primer, theforward outer primer, the backward outer primer, or any combinationthereof.

In some aspects, the plurality of primers further comprises a loopforward primer. In some aspects, the plurality of primers furthercomprises a loop backward primer. In some aspects, the loop forwardprimer is between an F1c region and an F2c region. In some aspects, theloop backward primer is between a B1c region and a B2c region.

In some aspects, the target nucleic acid comprises a single nucleotidepolymorphism (SNP). In some aspects, the single nucleotide polymorphism(SNP) comprises a HERC2 SNP. In some aspects, the single nucleotidepolymorphism (SNP) is associated with an increased risk or decreasedrisk of cancer. In some aspects, the target nucleic acid comprises asingle nucleotide polymorphism (SNP), and wherein the detectable signalis higher in the presence of a guide nucleic acid that is 100%complementary to the target nucleic acid comprising the singlenucleotide polymorphism (SNP) than in the presence of a guide nucleicacid that is less than 100% complementary to the target nucleic acidcomprising the single nucleotide polymorphism (SNP).

In some aspects, the plurality of primers and the guide nucleic acid arepresent together in a sample comprising the target nucleic acid. In someaspects, the amplifying and the contacting the sample to the guidenucleic acid occurs at the same time. In other aspects, the amplifyingand the contacting the sample to the guide nucleic acid occur atdifferent times. In some aspects, the method further comprises providinga polymerase, a dATP, a dTTP, a dGTP, a dCTP, or any combinationthereof.

The present disclosure provides an amplification method for inserting aPAM sequence into a target nucleic acid.

In some aspects, the present disclosure provides a method of assayingfor a target nucleic acid segment in a sample, wherein the targetnucleic acid segment lacks a PAM sequence, comprising amplifying thetarget nucleic acid segment using a primer having a region that isreverse complementary to the target nucleic acid segment and a regionthat has a PAM sequence reverse complement, thereby generating a PAMtarget nucleic acid having a PAM sequence adjacent to target sequence ofan amplification product; contacting the PAM target nucleic acid toPAM-dependent sequence specific nuclease complex comprising a guidenucleic acid and a programmable nuclease that exhibits sequenceindependent cleavage upon forming a complex comprising the segment ofthe guide nucleic acid binding to the segment of the PAM target nucleicacid; and assaying for cleavage of at least one detector nucleic acid ofa population of detector nucleic acids, wherein the cleavage indicates apresence of the target nucleic acid in the sample and wherein theabsence of the cleavage indicates an absence of the target nucleic acidin the sample. Often, the PAM comprises a sequence encoding dUdUdUN.Sometimes the PAM comprises a sequence encoding TTTN. The programmablenuclease is, for example, Cas12. The present disclosure further providesthe number of nucleotides in a nucleotide extension of the forwardprimer used to produce the PAM target nucleic acid, as well as thelocation of the mutation or mismatch in the PAM target nucleic acid.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The novel features of the disclosure are set forthwith particularity in the appended claims. A better understanding of thefeatures and advantages of the present disclosure will be obtained byreference to the following detailed description that sets forthillustrative embodiments, in which the principles of the disclosure areutilized, and the accompanying drawings of which:

FIG. 1 shows an improved SNP detection enzyme and method. At left isshown ALDH2 E540K G-SNP, while at right one sees ALDH E540K A-SNP. TheALDH2 G-SNP was detected with a G-SNP gRNA (SEQ ID NO: 425,UAAUUUCUACUAAGUGUAGAUACUUCAGUGUAUGCCUGCAG), and the ALDH2 A-SNP wasdetected with an A-SNP gRNA (SEQ ID NO: 426,UAAUUUCUACUAAGUGUAGAUACUUUAGUGUAUGCCUGCAG). LbCas12a (SEQ ID NO: 1) isshown at top, while a representative improved enzyme, a Cas12 variantcorresponding to (SEQ ID NO: 11), is shown at bottom.

FIG. 2 shows the first of a series of experiments to assess buffercontents for detection using a Cas12 variant (SEQ ID NO: 11).

FIG. 3 shows improvements conveyed by inclusion of acetate atconcentrations of about 0, 10, 20, 37, 75, 150, 300 and 600 mM, fromleft to right on detection using a Cas12 variant (SEQ ID NO: 11).

FIG. 4 shows an improvement in SNP specificity upon inclusion of heparinin a reaction buffer when detected with a Cas12 variant (SEQ ID NO: 11).

FIG. 5 shows optimization for a number of buffer additives, such asheparin, DTT, NP-40, and BSA (from left to right) over a series of 8iterative dilutions when detected with a Cas12 variant (SEQ ID NO: 11)and a gRNA of SEQ ID NO: 423.

FIG. 6 shows base sensitivity for alleles having bases A, C, G, or T, ata SNP position, for LbCas12a (SEQ ID NO: 1), top, and a representativeimproved enzyme, a Cas12 variant corresponding to (SEQ ID NO: 11),below. Target sequences corresponding to SEQ ID NO: 431-SEQ ID NO: 438were detected.

The A SNP allele was detected using a gRNA of SEQ ID NO: 427(GUUUGGUACCUUUAUUAAUUUCUACUAAGUGUAGAUGGCAGGCCAAACU GCUGGGU).The C SNP allele was detected using a gRNA of SEQ ID NO: 428(GUUUGGUACCUUUAUUAAUUUCUACUAAGUGUAGAUGGCCGGCCAAACU GCUGGGU).The G SNP allele was detected using a gRNA of SEQ ID NO: 429(GUUUGGUACCUUUAUUAAUUUCUACUAAGUGUAGAUGGCGGGCCAAACU GCUGGGU).The T SNP allele was detected using a gRNA of SEQ ID NO: 430(GUUUGGUACCUUUAUUAAUUUCUACUAAGUGUAGAUGGCUGGCCAAACU GCUGGGU).

FIG. 7 shows template optimization for an improved enzyme, a Cas12variant corresponding to SEQ ID NO: 11, as disclosed herein. Templatescomprising a C SNP allele (SEQ ID NO: 440, GGGCATGAGCTGCGTGATGA) or a TSNP allele (SEQ ID NO: 441, GGGCATGAGCTGCATGATGA) were detected usinggRNAs directed to the C SNP (SEQ ID NO: 423) or the T SNP allele (SEQ IDNO: 439, UAAUUUCUACUAAGUGUAGAUUCAUCAUGCAGCUCAUGCCC). Primerscorresponding to SEQ ID NO: 396 and SEQ ID NO: 397 were used to amplifythe target sequence and insert a PAM sequence.

FIG. 8 shows base sensitivity of an improved enzyme, a Cas12 variantcorresponding to SEQ ID NO: 11, for each allele having bases A, C, G, orT, for an EGFR SNP as disclosed herein. EGFR target sequencescorresponding to SEQ ID NO: 444-SEQ ID NO: 447 were detected. Primerscorresponding to SEQ ID NO: 442 (ACCACATGCAGGAAGGTCAG) and SEQ ID NO:443 (AGAAGGACTCCATTGCTGC) were used to amplify the target sequences. TheA SNP allele was detected using a gRNA of SEQ ID NO: 427. The C SNPallele was detected using a gRNA of SEQ ID NO: 428. The G SNP allele wasdetected using a gRNA of SEQ ID NO: 429. The T SNP allele was detectedusing a gRNA of SEQ ID NO: 430.

FIG. 9 shows an assessment of buffer additives and their effect ondetection using a Cas12 variant (SEQ ID NO: 11).

FIG. 10 shows trans cleavage activity of various Cas12 orthologs orother improved enzymes corresponding to SEQ ID NO: 586, SEQ ID NO: 581,SEQ ID NO: 576, SEQ ID NO: 587, SEQ ID NO: 578, SEQ ID NO: 572, SEQ IDNO: 575, SEQ ID NO: 11, SEQ ID NO: 573, SEQ ID NO: 589, and SEQ ID NO:583, and of LbCas12a (SEQ ID NO: 1) on targets containing various PAMs,double and single mismatched substrates. Target dsDNA was obtained byannealing complementary ssDNA primers with 2:1 ratio of non-targetstrand to target strand in hybridization buffer (50 mM NaCl, 1 mM TrispH 8.0, 0.1 mM EDTA) This ensures double-stranded DNA is being detectedinstead of single-stranded DNA. PAM sequences and the sequences of thetarget and non-target strands are provided in TABLE 29.

FIG. 11 shows trans cleavage activity of various Cas12 orthologs orother improved enzymes corresponding to SEQ ID NO: 2, SEQ ID NO: 1, SEQID NO: 590-SEQ ID NO: 598, SEQ ID NO: 580, and SEQ ID NO: 599-SEQ ID NO:602 on targets containing various PAMs, double and single mismatchedsubstrates. PAM sequences and the sequences of the target and non-targetstrands are provided in TABLE 29.

FIG. 12 shows trans cleavage activity of various Cas12 orthologs orother improved enzymes corresponding to SEQ ID NO: 571-SEQ ID NO: 577,SEQ ID NO: 11, SEQ ID NO: 578-SEQ ID NO: 589, SEQ ID NO: 1, and SEQ IDNO: 3 on targets containing various PAMs, double and single mismatchedsubstrates. PAM sequences and the sequences of the target and non-targetstrands are provided in TABLE 29. Figure discloses SEQ ID NOS 381-393,respectively, in order of appearance.

FIG. 13A, FIG. 13B, and FIG. 13C show trans cleavage activity of variousCas12 orthologs corresponding to SEQ ID NO: 571-SEQ ID NO: 577, SEQ IDNO: 11, SEQ ID NO: 578-SEQ ID NO: 589, SEQ ID NO: 1, and SEQ ID NO: 3 onPCR targets containing a TTTA (SEQ ID NO: 384) PAM using various guideRNA repeat sequences. Activity was detected in the presence of differentCas12 variants and different pre-crRNAs corresponding to different Cas12variants. Sequences of the pre-crRNAs are provided in TABLE 30.

FIG. 14 shows activity of various Cas12 orthologs and other improvedenzymes corresponding to SEQ ID NO: 571-SEQ ID NO: 577, SEQ ID NO: 11,SEQ ID NO: 578-SEQ ID NO: 589, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:590-SEQ ID NO: 598, SEQ ID NO: 580, SEQ ID NO: 599-SEQ ID NO: 602, andSEQ ID NO: 2 on a target PCR product. The negative control (“(−)control”) is PCR product with no Cas12 added. The positive control iscleavage with a BamHI restriction enzyme (“BamHI”). Numbers above eachlane correspond to the time in minutes before the reaction was quenchedwith 10 mM EDTA. Lanes marked with “−” under each Cas12 orthologcorrespond to negative control conditions with protein but no crRNA.

FIG. 15 shows limit of detection (LOD) assay results indicating transcleavage activity of various Cas12 orthologs or other improved enzymescorresponding to SEQ ID NO: 572, SEQ ID NO: 576, SEQ ID NO: 11, SEQ IDNO: 582, SEQ ID NO: 583, SEQ ID NO: 587, SEQ ID NO: 1, SEQ ID NO: 591,SEQ ID NO: 595, SEQ ID NO: 597, SEQ ID NO: 600, SEQ ID NO: 601, and SEQID NO: 2 in the presence of various activator concentrations (shown onthe left).

FIG. 16A and FIG. 16B show trans cleavage activity of various Cas12orthologs corresponding to SEQ ID NO: 590-SEQ ID NO: 598, SEQ ID NO:580, SEQ ID NO: 599-SEQ ID NO: 602, and SEQ ID NO: 2 in the presence ofvarious salt concentrations.

FIG. 17A and FIG. 17B show trans cleavage activity of various Cas12orthologs corresponding to SEQ ID NO: 590-SEQ ID NO: 598, SEQ ID NO:580, and SEQ ID NO: 599-SEQ ID NO: 602, and SEQ ID NO: 2 in the presenceof various salt concentrations.

FIG. 18 shows activity of three programmable nucleases, a Cas12 variant(SEQ ID NO: 11), LbCas12a (SEQ ID NO: 1), and LbuCas13a (SEQ ID NO: 104,also referred to herein as Lbu C2C2). The results show that thefunctional range for the Cas12 variant (SEQ ID NO: 11) is between 25° C.and 45° C., with maximal activity at 35° C.

FIG. 19 shows the results of incubating two Cas12 proteins, SEQ ID NO: 1and SEQ ID NO: 11, for 15 minutes at 45° C., 50° C., 55° C., 60° C., 65°C., or 70° C. and then decreasing the reaction temperature to 37° C.

FIG. 20 shows that the stability of a Cas12 variant (SEQ ID NO: 11) atelevated temperatures is dependent on the buffer composition.

FIG. 21 shows graphs of activity of a Cas13 (SEQ ID NO: 104), asmeasured by fluorescence, with (left graph) and without (right graph)activator over time.

FIG. 22 shows inhibition of Cas13a (SEQ ID NO: 104) activity by SDS andurea.

FIG. 22A shows the Cas13a (SEQ ID NO: 104) detection assay performed inthe presence of 0-200 mM urea.

FIG. 22B shows complete inhibition of Cas13a (SEQ ID NO: 104) uponaddition of 0.1% or greater amounts of SDS to the reaction (left graphshows with activator and right graph shows without activator).

FIG. 23 shows the performance of Cas13a (SEQ ID NO: 104) in DETECTRreactions with varying concentrations of salt.

FIG. 23A shows the results of varying the concentration of NaCl in aCas13a (SEQ ID NO: 104) DETECTR reaction.

FIG. 23B shows the results of varying the concentration of KCl in aCas13a (SEQ ID NO: 104) DETECTR reaction.

FIG. 24 shows optimization of DTT concentration in a Cas13a (SEQ ID NO:104) DETECTR assay.

FIG. 24A shows activity of a Cas13a (SEQ ID NO: 104) at varying DTTconcentration in NaCl.

FIG. 24B shows activity of a Cas13a (SEQ ID NO: 104) at varying DTTconcentrations in KCl. The orange bar indicates buffer conditions with50 mM KCl and no DTT. In addition to the indicated KCl and DTTconcentration, each buffer condition also contained 20 mM HEPES pH 6.8,5 mM MgCl₂, 10 μg/mL BSA, 100 μg/mL tRNA, 0.01% Igepal Ca-630 (NP-40),and 5% Glycerol).

FIG. 25 shows the activity of Cas13a (SEQ ID NO: 104) in the DETECTRassay, as measured by fluorescence, for each of the tested reporters.

FIG. 26 shows Cas13a (SEQ ID NO: 104) activity in the DETECTR assay, asmeasured by fluorescence, for each of the tested conditions.

FIG. 27 shows Cas13a (SEQ ID NO: 104) performance in the DETECTR assay,as measured by fluorescence, for each of the five commercially availablebuffers and a HEPES pH 6.8 buffer (“Normal,” 20 mM HEPES pH 6.8; 50 mMKCl; 5 mM MgCl₂; 10 μg/mL BSA; 100 μg/mL tRNA; 0.01% Igepal Ca-630(NP-40); 5% Glycerol).

FIG. 28 shows a comparison of the a HEPES pH 6.8 buffer (“OriginalBuffer,” 20 mM HEPES pH 6.8; 50 mM KCl; 5 mM MgCl₂; 10 μg/mL BSA; 100μg/mL tRNA; 0.01% Igepal Ca-630 (NP-40); 5% Glycerol) to an highperformance buffer (“MBuffer1,” 20 mM imidazole pH 7.5, 50 mM KCl, 5 mMMgCl₂, 10 μg/μL BSA, 0.01% Igepal Ca-630, and 5% glycerol) for a Cas13a(SEQ ID NO: 104) DETECTR assay with serially diluted target RNAs and runat 37° C. for 30 minutes.

FIG. 29 shows that 5% glycerol in an high performance buffer(“MBuffer1,” left graph, 20 mM imidazole pH 7.5, 50 mM KCl, 5 mM MgCl₂,10 μg/μL BSA, 0.01% Igepal Ca-630, and 5% glycerol) increasesperformance of a Cas13a (SEQ ID NO: 104) DETECTR assay in comparison toan identical buffer without glycerol (right graph).

FIG. 30 shows a gradient chart of Cas13a (SEQ ID NO: 104) activity inthe DETECTR assay, as measured by fluorescence, (darker squares indicateincreased Cas13a activity) versus varying NP-40 concentration along thex-axis and varying BSA concentration along the y-axis. In addition tothe indicated concentrations of NP-40 and BSA, each buffer contained 20mM imidazole pH 7.5, 50 mM KCl, 5 mM MgCl₂, and 5% glycerol.

FIG. 31 shows Cas13a (SEQ ID NO: 104) performance in DETECTR assays, asmeasured by fluorescence, versus the different additives tested.

FIG. 32 shows the results of screening 84 different buffer and pHcombinations to determine the optimal buffer for LbCas12a (SEQ ID NO: 1)activity in DETECTR assays, as measured by fluorescence.

FIG. 33 shows LbCas12a (SEQ ID NO: 1) performance in DETECTR assays, asmeasured by fluorescence, in each of the tested conditions.

FIG. 34 shows a Cas12 variant (SEQ ID NO: 11) performance in DETECTRassays, as measured by fluorescence, for each of the tested conditions(buffer type and pH).

FIG. 35 shows a Cas12 variant (SEQ ID NO: 11) performance in DETECTRassays, as measured by fluorescence, for the various salt types andconcentrations tested.

FIG. 36 shows a Cas12 variant (SEQ ID NO: 11) performance in DETECTRassays, as measured by fluorescence (darker squares indicate greaterfluorescence and more activity), versus heparin concentration on thex-axis and KOAc buffer concentration on the y-axis.

FIG. 37 shows that specific compounds inhibited the performance of theCas12 variant (SEQ ID NO: 11) DETECTR assay including: benzamidinehydrochloride, beryllium sulfate, manganese chloride, potassium bromide,sodium iodine, zinc chloride, di-ammonium hydrogen phosphate,tri-lithium citrate, tri-sodium citrate, cadmium chloride, copperchloride, yttrium chloride, 1-6 diaminohexane, 1-8-diaminooctane,ammonium fluoride, and ammonium sulfate.

FIG. 38 shows the results of evaluating SNP sensitivity along targetsequences for a Cas12 variant (SEQ ID NO: 11). Figure discloses SEQ IDNOS 416 and 417, respectively, in order of appearance.

FIG. 39 shows the results of evaluating SNP sensitivity along targetsequences for a Cas12 variant (SEQ ID NO: 11). Figure discloses SEQ IDNOS 416 and 417, respectively, in order of appearance.

FIG. 40 shows schemes for designing primers for loop mediated isothermalamplification (LAMP) of a target nucleic acid sequence. Regions denotedby “c” are reverse complementary to the corresponding region not denotedby “c” (e.g., region F3c is reverse complementary to region F3).

FIG. 41A, FIG. 41B, FIG. 41C, and FIG. 41D show schematics of exemplaryconfigurations of various regions of a nucleic acid sequence thatcorrespond to or anneal LAMP primers or guide RNA sequences, or thatcomprise protospacer-adjacent motif (PAM) or protospacer flanking site(PFS), and target nucleic acid sequences for amplification and detectionby LAMP and DETECTR.

FIG. 41A shows a schematic of an exemplary arrangement of the guide RNA(gRNA) with respect to the various regions of the nucleic acid sequencethat correspond to or anneal LAMP primers. In this arrangement, theguide RNA is reverse complementary to a sequence of the target nucleicacid, which is between an F1c region (a region reverse complementary toan F1 region) and a B1 region.

FIG. 41B shows a schematic of an exemplary arrangement of the guide RNAsequence with respect to the various regions of the nucleic acidsequence that correspond to or anneal LAMP primers. In this arrangement,the guide RNA is partially reverse complementary to a sequence of thetarget nucleic acid, which is between an F1c region and a B1 region. Forexample, the target nucleic acid comprises a sequence between an F1cregion and a B1 region that is reverse complementary to at least 60% ofa guide nucleic acid. In this arrangement, the guide RNA is not reversecomplementary to the forward inner primer or the backward inner primershown in FIG. 40.

FIG. 41C shows a schematic of an exemplary arrangement of the guide RNAwith respect to the various regions of the nucleic acid sequence thatcorrespond to or anneal LAMP primers. In this arrangement, the guide RNAhybridizes to a sequence of the target nucleic acid, which is within theloop region between the B1 region and the B2 region. The primersequences do not contain and are not reverse complementary to the PAM orPFS.

FIG. 41D shows a schematic of an exemplary arrangement of the guide RNAwith respect to the various regions of the nucleic acid sequence thatcorrespond to or anneal LAMP primers. In this arrangement, the guide RNAhybridizes to a sequence of the target nucleic acid, which is within theloop region between the F2c region and F1c region. The forward innerprimer, backward inner primer, forward outer primer, and backward outerprimer sequences do not contain and are not reverse complementary to thePAM or PFS.

FIG. 42A, FIG. 42B, and FIG. 42C show schematics of exemplaryconfigurations of various regions of the nucleic acid sequence thatcorrespond to or anneal LAMP primers or guide RNA sequences, or compriseprotospacer-adjacent motif (PAM) or protospacer flanking site (PFS), andtarget nucleic acid sequences for combined LAMP and DETECTR foramplification and detection, respectively. At the right, the schematicsalso show corresponding fluorescence data using the LAMP amplificationand guide RNA sequences to detect the presence of a target nucleic acidsequence, where a fluorescence signal is the output of the DETECTRreaction and indicates presence of the target nucleic acid.

FIG. 42A shows a schematic of an arrangement of various regions of thenucleic acid sequence that correspond to or anneal LAMP primers (SEQ IDNO: 201, SEQ ID NO: 202, SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO:249-SEQ ID NO: 252) and positions of three guide RNAs (gRNA1 (SEQ ID NO:261), gRNA2 (SEQ ID NO: 262), and gRNA3 (SEQ ID NO: 263)) relative tothe LAMP primers (at left). gRNA1 overlaps with the B2c region and is,thus, reverse complementary to the B2 region. gRNA2 overlaps with the B1region and is, thus, reverse complementary to the B1c region. gRNA3partially overlaps with the B3 region and partially overlaps with the B2region and is, thus, partially reverse complementary to the B3c regionand partially reverse complementary to the B2c region. The complementaryregions (B1c, B2c, B3c, F1c, F2c, and F3c) are not depicted, butcorrespond to the regions shown in FIG. 40. At right is a graph offluorescence from the DETECTR reaction in the presence of 10,000 genomecopies (before amplification) of the target nucleic acid or 0 genomecopies of the target nucleic acid.

FIG. 42B shows a schematic of an arrangement of various regions of thenucleic acid sequence that correspond to or anneal LAMP primers (SEQ IDNO: 202, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 214, SEQ ID NO: 215,SEQ ID NO: 253-SEQ ID NO: 255) and positions of three guide RNAs (gRNA1(SEQ ID NO: 261), gRNA2 (SEQ ID NO: 262), and gRNA3 (SEQ ID NO: 263))relative to the LAMP primers (at left). gRNA1 overlaps with the B1cregion and is, thus, reverse complementary to the B1 region. gRNA2overlaps with the LF region and is, thus, reverse complementary to theLFc region. gRNA 3 partially overlaps with the B2 region and partiallyoverlaps with the LBc region and is, thus, partially reversecomplementary to the B2c region and is partially reverse complementaryto the LB region. At right is a graph of fluorescence from the DETECTRreaction in the presence of 10,000 genome copies (before amplification)of the target nucleic acid or 0 genome copies of the target nucleicacid.

FIG. 42C shows a schematic of an arrangement of various regions of thenucleic acid sequence that correspond to or anneal LAMP primers (SEQ IDNO: 184, SEQ ID NO: 188, SEQ ID NO: 255-SEQ ID NO: 260) and positions ofthree guide RNAs (gRNA1 (SEQ ID NO: 261), gRNA2 (SEQ ID NO: 262), andgRNA3 (SEQ ID NO: 263)) relative to the LAMP primers (at left). gRNA1overlaps with the B1c region and is, thus, reverse complementary to theB1 region. gRNA2 partially overlaps with the LF region and partiallyoverlaps with the F2c region and is, thus, partially reversecomplementary to the LFc region and partially reverse complementary tothe F2 region. gRNA3 overlaps with the B2 and is, thus, reversecomplementary to the B2c region. At right is a graph of fluorescencefrom the DETECTR reaction in the presence of 10,000 genome copies(before amplification) of the target nucleic acid or 0 genome copies ofthe target nucleic acid.

FIG. 43A shows a detailed breakdown of the arrangement and sequences ofvarious regions of the nucleic acid sequence that correspond to oranneal LAMP primers or guide RNA sequences, or compriseprotospacer-adjacent motif (PAM) or protospacer flanking site (PFS), andtarget nucleic acid sequences for the LAMP and DETECTR assays shown inFIG. 42A.

FIG. 43B shows a detailed breakdown of the arrangement and sequences ofvarious regions of the nucleic acid sequence that correspond to oranneal LAMP primers or guide RNA sequences, or compriseprotospacer-adjacent motif (PAM) or protospacer flanking site (PFS), andtarget nucleic acid sequences for the LAMP and DETECTR assays shown inFIG. 42B.

FIG. 43C shows a detailed breakdown of the arrangement and sequences ofvarious regions of the nucleic acid sequence that correspond to oranneal LAMP primers or guide RNA sequences, or compriseprotospacer-adjacent motif (PAM) or protospacer flanking site (PFS), andtarget nucleic acid sequences for the LAMP and DETECTR assays shown inFIG. 42C.

FIG. 44 shows the time to result of a reverse-transcription LAMP(RT-LAMP) reaction detected using a DNA binding dye. Amplification wasperformed using primer sets #1-#10. Sequences of the primer sets areprovided in TABLE 10.

FIG. 45 shows fluorescence signal from a DETECTR reaction following afive-minute incubation with products from RT-LAMP reactions.Amplification was performed using primer sets #1-#10. Sequences of theprimer sets are provided in TABLE 10. LAMP primer sets #1-6 weredesigned for use with guide RNA #2 (SEQ ID NO: 240), and LAMP primersets #7-10 were designed for use with guide RNA #1 (SEQ ID NO: 239).

FIG. 46 shows detection of sequences from influenza A virus (IAV) usingSYTO 9 (a DNA binding dye) following RT-LAMP amplification with LAMPprimer sets 1, 2, 4, 5, 6, 7, 8, 9, 10, or a negative control. Sequencesof the primer sets are provided in TABLE 12.

FIG. 47 shows the time to amplification of an influenza B virus (IBV)target sequence following RT-LAMP amplification. Amplification wasdetected using SYTO 9 in the presence of increasing concentrations oftarget sequence (0, 100, 1000, 10,000, or 100,000 genome copies of thetarget sequence per reaction).

FIG. 48 shows the time to amplification of an IAV target sequencefollowing LAMP amplification with different primer sets.

FIG. 49 shows detection of target nucleic acid sequences from influenzaA virus (IAV) using DETECTR following RT-LAMP amplification with LAMPprimer sets 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, or a negative control. Tenreactions were performed per primer set. DETECTR signal was measured asa function of an amount of target sequence present in the reaction.Sequences of the primer sets are provided in TABLE 12.

FIG. 50 shows a scheme for designing primers for LAMP amplification of atarget nucleic acid sequence and detection of a single nucleotidepolymorphism (SNP) in the target nucleic acid sequence. In an exemplaryarrangement, the SNP of the target nucleic acid is positioned betweenthe F1c region and the B1 region.

FIG. 51 shows schematics of exemplary arrangements of LAMP primers,guide RNA sequences, protospacer-adjacent motif (PAM) or protospacerflanking site (PFS), and target nucleic acids with a SNP for methods ofLAMP amplification of a target nucleic acid and detection of the targetnucleic acid using DETECTR.

FIG. 51A shows a schematic of an exemplary arrangement of the guide RNAwith respect to various regions of the nucleic acid sequence thatcorrespond to or anneal LAMP primers. In this arrangement, the PAM orPFS of the target nucleic acid is positioned between an F1c region and aB1 region. The entirety of the guide RNA sequence may be between the F1cregion and the B1c region. The SNP is shown as positioned within asequence of the target nucleic acid that hybridizes to the guide RNA.

FIG. 51B shows a schematic of an exemplary arrangement of the guide RNAsequence with respect to various regions of the nucleic acid sequencethat correspond to or anneal LAMP primers. In this arrangement, the PAMor PFS of the target nucleic acid is positioned between an F1c regionand a B1 region and the target nucleic acid comprises a sequence betweenan F1c region and a B1 region that is reverse complementary to at least60% of a guide nucleic acid. In this example, the guide RNA is notreverse complementary to the forward inner primer or the backward innerprimer. The SNP is shown as positioned within a sequence of the targetnucleic acid that hybridizes to the guide RNA.

FIG. 51C shows a schematic of an exemplary arrangement of the guide RNAsequence with respect to various regions of the nucleic acid sequencethat correspond to or anneal LAMP primers. In this arrangement, the PAMor PFS of the target nucleic acid is positioned between the F1c regionand the B1 region and the entirety of the guide RNA sequence is betweenthe F1c region and the B1 region. The SNP is shown as positioned withina sequence of the target nucleic acid that hybridizes to the guide RNA.

FIG. 52 shows an exemplary sequence of a nucleic acid comprising two PAMsites and a HERC2 SNP. The positions of two gRNAs targeting the HERC2 ASNP allele at either position 9 with respect to a first PAM site (SEQ IDNO: 245) or at position 14 with respect to a second PAM site (SEQ ID NO:247) are shown. The position of a SNP is indicated with a triangle.

FIG. 53 shows results from DETECTR reactions to detect a HERC2 SNP atposition 9 with respect to a first PAM site or position 14 with respectto a second PAM site following LAMP amplification. Fluorescence signal,indicative of detection of the target sequence, was measured over timein the presence of a target sequence comprising either a G allele or anA allele in HERC2. The target sequence was detected using a guide RNA(crRNA only) to detect either the A allele or the G allele (SEQ ID NO:245-SEQ ID NO: 248).

FIG. 54 shows a heatmap of fluorescence from a DETECTR reactionfollowing LAMP amplification of the target nucleic acid sequence. TheDETECTR reaction differentiated between two HERC2 SNP alleles atposition 9 with respect to the PAM, using guide RNAs (crRNA only)specific for the A allele (SEQ ID NO: 245, “R570 A SNP”) or the G allele(SEQ ID NO: 246, “R571 G SNP”). Positive detection is indicated by ahigh fluorescence value in the DETECTR reaction.

FIG. 55 shows combined LAMP amplification of a target nucleic acid byLAMP and detection of the target nucleic acid by DETECTR. Detection wascarried out visually with DETECTR by illuminating the samples with a redLED. Each reaction contained a target nucleic acid sequence comprising aSNP allele for either a blue eye phenotype (“Blue Eye”) or a brown eyephenotype (“Brown Eye”). Samples “Brown*” and “Blue*” were an A allelepositive control and a G allele positive control, respectively. Aposition 9 guide RNA for either the brown eye phenotype (SEQ ID NO: 245,“Br”) or the blue eye phenotype (SEQ ID NO: 246, “B1”) was used for eachLAMP DETECTR reaction.

FIG. 56A, FIG. 56B, FIG. 56C, FIG. 56D, FIG. 56E, FIG. 56F, FIG. 56G,and FIG. 56H show high sensitivity and high specificity buffers forLbCas12a (SEQ ID NO: 1). In the presence of 50 μg/ml heparin and 100 mMsalt, LbCas12a has improved targeting specificity and enhanced SNPdiscrimination capabilities. Target sequences were detected using acrRNA directed to the EGFR wild type sequence (SEQ ID NO: 448,UAAUUUCUACUAAGUGUAGAUGGCUGGCCAAACUGCUGGGU) or a crRNA directed to theEGFR mutant sequence (G SNP, SEQ ID NO: 449,UAAUUUCUACUAAGUGUAGAUGGCGGGCCAAACUGCUGGGU). In the absence of heparinand salt, Cas12a has improved sensitivity. For all SNP-related studies,high specificity buffer was used.

FIG. 57 shows a schematic of PCR primers and guide RNA targetingsequence for EGFR T790M SNP. The forward primer represents a“PAMplification primer” (SEQ ID NO: 396), which embeds a PAM sequence(‘TTTV’) upstream of the targeting sequence and includes a 6 nt 3′extension for priming. The PAM sequence is required for Cas12a-gRNA torecognize the matching DNA target. In this schematic, the guide RNA isdesigned to target the mismatch located 7 nucleotides (nt) downstream ofthe 5′ end of the target sequence (SEQ ID NO: 400). This guideRNA/primer design is used for FIG. 59-FIG. 61.

FIG. 58A, FIG. 58B, and FIG. 58C show the PAM forward (F) primer (alsoreferred to as a PAMplification primer) used in amplification. PAM Fprimers with varying 3′ extensions (4 nt, 5 nt, 6 nt, SEQ ID NO: 394,SEQ ID NO: 395, and SEQ ID NO: 396, respectively) were tested with guideRNA targeting T790M with mismatch at the 7^(th) position (SEQ ID NO:400). The PAM F primer with 6 nt extension (SEQ ID NO: 396) demonstratedoptimal detection with the guide RNA. This PAM F primer was used forFIG. 60-FIG. 63. The PAMplification primer produces dU-containingamplicons for detection of mutant sequences at low frequency. Cas12guide RNAs were designed to target the T790M mutant allele (c.2369C>T,at guide mismatch position 7) in Horizon Discovery EGFR cfDNA standardsat 0-5% minor allele frequencies (MAF) with 2 ng input DNA.PAMplification primers include 4-6 nt extensions at the 3′ enddownstream of the embedded PAM. n=3 technical replicates; bars representmean±SD.

FIG. 59A-FIG. 59C illustrate that Cas12 guide RNAs designed to target awild type sequence (“WT” C SNP allele) and sequence comprising a T790M TSNP allele show specific Cas12-based detection in the presence ofcognate single nucleotide polymorphism (SNP). Targets were detected witha crRNA directed to the wild type sequence (SEQ ID NO: 423) or a crRNAdirected to the T SNP allele sequence (SEQ ID NO: 439). Time coursesshow activation of the WT or mutant crRNA only in the presence of thematching target (FIG. 59A and FIG. 59B). Heatmap represents time coursedata at t=60 min (FIG. 4C) n=3 technical replicates; synthetic oligotargets; bars represent mean±SD.

FIG. 60A-FIG. 60D show Cas12a can detect down to 0.1-1% minor allelefrequency (MAF) of EGFR T790M (T SNP allele) in mock cfDNA samples(Horizon Discovery), with 2 ng total DNA input and a PCRpre-amplification step. Targets were detected with a crRNA directed tothe wild type sequence (SEQ ID NO: 423) or a crRNA directed to the T SNPallele sequence (SEQ ID NO: 439). Detection of WT (C SNP allele) andmutant allele at t=90 min with low frequency EGFR standards (FIG. 60A).Bar graphs of mutant allele detection only (FIG. 60B). Heatmaprepresentation of WT and mutant allele detection (FIG. 60C). Thedetection of low frequency SNPs using PAMplification with 6 nt extensionand dU-containing amplicons. Cas12a can detect down to 0.1-1% minorallele frequency (MAF) of EGFR T790M in mock cfDNA samples (HorizonDiscovery), with 2 ng total DNA input. n=3 replicates, two-tailedStudent's t-test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; barsrepresent mean plus SD. FIG. 60D shows the different percentages of theWT and mutant allele in sample in a single test tube as pictorialrepresentation of the percentage of MAF in the samples tested.

FIG. 61 shows limit of detection studies illustrating that 2 ng totalDNA is the minimum input allowed for detection of 0.1-1% minor allelefrequency (MAF) of EGFR T790M (T SNP allele) in mock cfDNA samples(Horizon Discovery) with a PCR pre-amplification step. n=3 replicates,two-tailed Student's t-test; *p<0.05, **p<0.01, ***p<0.001,****p<0.0001; bars represent mean plus SD. Targets were detected using acrRNA directed to T SNP allele (SEQ ID NO: 403). Targets were amplifiedusing primers corresponding to SEQ ID NO: 396 and SEQ ID NO: 397.

FIG. 62 shows a table of FIG. 61 assay parameters.

FIG. 63A-FIG. 63D show a blocking primer strategy.

FIG. 63A shows how the blocking primer blocks the forward primer frombinding to the WT nucleic acid for amplification.

FIG. 63B shows how the mutation in SNP does not result in the binding ofthe blocking primer, and therefore allowing the forward primer to bindto the SNP nucleic acid for amplification.

FIG. 63C and FIG. 63D show the detection of the EGFR C SNP using aninput of 6 ng and the detection of the EGFR T SNP using an input of 6ng, respectively, after amplification using the blocking primer strategyof FIG. 64A and FIG. 64B. PAMplification and blocking primers areprovided in TABLE 16.

FIG. 64A-FIG. 64B and FIG. 65A-FIG. 65B illustrate COLD-PCR strategies.FIG. 64A shows a full COLD-PCR strategy. FIG. 64B shows a fast COLD-PCRstrategy.

FIG. 65A shows the detection of the EGFR C SNP using an input of 6 ngand a crRNA corresponding to SEQ ID NO: 423 and LbCas12a (SEQ ID NO: 1)after amplification using COLD-PCR. FIG. 65B shows the detection of theEGFR C SNP using an input of 6 ng the detection of the EGFR T SNP usingan input of 6 ng and a crRNA corresponding to SEQ ID NO: 439 andLbCas12a (SEQ ID NO: 1) after amplification using COLD-PCR. COLD-PCR wasperformed using primers corresponding to SEQ ID NO: 396 and SEQ ID NO:397.

FIG. 66A-FIG. 66B show experimental data illustrating that LbCas12a (SEQID NO: 1) can detect as low as 0.1% minor allele frequency (MAF) of EGFRL858R G SNP allele in synthetic DNA samples (Gblock), with a 1 nM totalDNA input and a cold-PCR pre-amplification step. FIG. 66A showsdetection of the mutant allele using a gRNA corresponding to SEQ ID NO:429 and FIG. 66B shows detection of the WT allele using a gRNAcorresponding to SEQ ID NO: 430 at t=40 minutes. (n=3 replicates,two-tailed Student's t-test; bars represent mean plus SD). Targetsequences were amplified using primers corresponding to SEQ ID NO: 450(GGCAGCCAGGAACGTACTG) and SEQ ID NO: 451 (CCTTCTGCATGGTATTCTTTCTCTTCC).

FIG. 67 shows the results of an EGFR exon 19 deletion Guide Screen usingLbCas12a (SEQ ID NO: 1). Twenty-six guides corresponding to SEQ ID NO:480-SEQ ID NO: 506 were designed and screened on 1 nM synthetic DNA(Twist fragments). Two guides (SEQ ID NO: 493 and SEQ ID NO: 499)yielded DETECTR signals similar to wild-type. The remaining 24 guidesshowed activity greater than wild-type with three standing out with thehighest DETECTR activity (SEQ ID NO: 485, SEQ ID NO: 488, and SEQ ID NO:490). Targets corresponding to SEQ ID NO: 452-SEQ ID NO: 477 and SEQ IDNO: 479 were detected. Target sequences are provided in TABLE 21.

FIG. 68A-FIG. 68B and FIG. 69A-FIG. 69B show the PAM forward primer(also referred to as a PAMplification primer). The single nucleotidemismatch was anchored at positions 3-8 or 5-8 nt downstream of the PAM.PAMplification primers with 2 nt or 4 nt extensions at the 3′ end weretested for their ability to discriminate the non-cognate targetcontaining a single nucleotide mismatch/polymorphisph (SNP). Here, a 4nt PAMplification 3′ extension is better at SNP detection compared tothe 2 nt extension. The mismatch position is optimal around positions 6(“6 nt mm”), 7 (“7 nt mm”) or 8 (“8 nt mm”). Primers used in this assayare provided in TABLE 22. Targets were detected using LbCas12a (SEQ IDNO: 1) and a gRNA corresponding to SEQ ID NO: 264(UAAUUUCUACUAAGUGUAGAUAACUUGACAUUUAAUGCUCA).

FIG. 70A-FIG. 70B show that Cas12 recognizes dU-containing PAM andtarget sequences from 100 nM to 10 pM. FIG. 70A: WT SNP-targeting guideRNA; FIG. 70B: mutant SNP-targeting guide RNA. Left to right for bothFIG. 70A and FIG. 70B: (top left) WT sequence with dT-containing target,(top middle) mutant sequence with dT-containing target, (top right)mutant sequence with dU-containing PAM and target, (bottom left) notarget, (bottom right) mutant sequence with dT-containing PAM anddU-containing target. Cas12 (SEQ ID NO: 1) is capable of SNP detectionwith dU-containing sequences (both PAM and target) without compromisingsensitivity. Primers used in this assay are provided in TABLE 22.

FIG. 71A-FIG. 71B show the detection of ALDH2 WT allele from humangenomic DNA (SEQ ID NO: 417) with dU-containing amplicons with Cas12.The ALDH2 gene was amplified from human saliva containing the WT alleleusing Taq master mix containing dUTP in place of dTTP, such that all Tnucleotides with the annotated ALDH2 target sequence shown in FIG. 71Ahave been replaced by U nucleotides. The amplicon was added directly toa Cas12 DETECTR assay. Cas12 guide RNAs targeting the ALDH2 WT alleledetected only the cognate WT sequence and not the mutant allele,demonstrating that Cas12 is capable of SNP detection with dU-containingtargets. Figure discloses SEQ ID NOS 752-753, respectively, in order ofappearance.

FIG. 71B shows a DETECTR reaction of an ALDH2 target nucleic acidsequence amplified with dUTPs using LbCas12a (SEQ ID NO: 1).Fluorescence was measured over time in the presences of the wild typenucleic acid sequence (“WT SNP”), a sequence with a point mutation(“Mutant SNP”), or a negative control without the target nucleic acidsequence.

FIG. 72 shows detection of amplified HERC2 genomic DNA using a Cas12variant (SEQ ID NO: 11) in the presence of increasing amounts of LAMPamplified DNA (“LAMP.Amplicon”). Each detection reaction was performedin the presence of 0 μL (negative control) of LAMP amplified DNA or from1 μL to 14 μL LAMP amplified DNA per 20 μL reaction.

FIG. 73 shows a schematic of addition of an artificial PAM to LAMP FIPor BIP primers. PAMs were introduced at different positions within theLAMP primer, and gRNAs were designed relative to each PAM for use inCRISPR-based detection assays of target nucleic acids.

FIG. 74 shows LAMP amplification of a target human genomic DNA (HERC2,SEQ ID NO: 416) with an FIP primer having PAM sequences at varyingpositions to introduce an artificial PAM in the HERC2 target nucleicacid. Amplification was monitored using a SYTO9 DNA binding dye. Thetarget was amplified using primers corresponding to SEQ ID NO: 233-SEQID NO: 234 and SEQ ID NO: 236-SEQ ID NO: 238 with a variable FIPdepending on the position of the artificially introduced PAM. FIPscorresponding to SEQ ID NO: 265-SEQ ID NO: 281 were used to insertartificial PAMs at position 1-position 17, respectively. The FIPcorresponding to SEQ ID NO: 235 was used to amplify the target withoutintroducing a PAM.

FIG. 75 shows detection of a target nucleic acid with an artificiallyintroduced PAM using a Cas12 variant (SEQ ID NO: 11). gRNAscorresponding to SEQ ID NO: 283-SEQ ID NO: 299 were used to detecttarget nucleic acids with artificially introduced PAMs at position1-position 17, respectively.

FIG. 76 shows detection of single point mutations at different positionsalong a nucleic acid sequence using a SEQ ID NO: 11 programmablenuclease. Point mutations corresponding to all possible nucleic acidswere inserted at different positions within either a HERC2 targetsequence (top, wild type sequence corresponding to SEQ ID NO: 416) or anALDH2 target sequence (bottom, wild type sequence corresponding to SEQID NO: 417). The HERC2 sequence was detected using a gRNA correspondingto SEQ ID NO: 246 (top plot) and the ALDH sequence was detected using agRNA corresponding to SEQ ID NO: 425 (bottom plot).

FIG. 77 shows detection of two PNPLA3 SNPs in a target nucleic acidsequence without a native PAM using a SEQ ID NO: 11 programmablenuclease. Guide RNAs corresponding to SEQ ID NO: 300-SEQ ID NO: 319 weredirected to the wild type (SEQ ID NO: 415, “WT”) sequence on the forwardstrand at position 1-position 20, respectively. gRNAs corresponding toSEQ ID NO: 320-SEQ ID NO: 339 were directed to the wild type (“WT”)sequence on the reverse strand at position 1-position 20, respectively.gRNAs corresponding to SEQ ID NO: 340-SEQ ID NO: 359 were directed tothe mutant (SEQ ID NO: 414, “rs738409”) sequence on the forward strandat position 1-position 20, respectively. gRNAs corresponding to SEQ IDNO: 360-SEQ ID NO: 379 were directed to the mutant (“rs738409”) sequenceon the reverse strand at position 1-position 20, respectively. Each gRNAwas used to detect four different target sequences corresponding to thewild type sequence (SEQ ID NO: 415, “WT”), a sequence with a pointmutation at a first site (SEQ ID NO: 413, “rs738408”), a sequence with apoint mutation at a second site (SEQ ID NO: 414, “rs738409”), or asequence with point mutations at both the first site and the second site(SEQ ID NO: 412, “rs738409+rs738408”).

FIG. 78 shows detection of single and double mutations in a targetnucleic acid sequence using a SEQ ID NO: 11 programmable nuclease.Target sequences corresponding to SEQ ID NO: 412-SEQ ID NO: 415 weredetected.

FIG. 79 shows detection of two PNPLA3 SNPs in a target nucleic acidsequence without a native PAM using a SEQ ID NO: 11 programmablenuclease. Target sequences corresponding to SEQ ID NO: 412-SEQ ID NO:415 were detected. A sample without a target sequence (non-targetcontrol, “NTC”) was used as a negative control. Sequences were detectedusing pooled gRNAs directed to either the wild type sequence (SEQ ID NO:301 and SEQ ID NO: 421, “WT DETECTR”) or the sequence containing amutation at the second position (SEQ ID NO: 341 and SEQ ID NO: 422,“rs738409 DETECTR”).

FIG. 80 shows detection of single point mutations at different positionsalong a nucleic acid sequence using LbCas12a (SEQ ID NO: 1). Pointmutations corresponding to all possible nucleic acids were inserted atdifferent positions within either a HERC2 target sequence (top, wildtype sequence corresponding to SEQ ID NO: 416) or an ALDH2 targetsequence (bottom, wild type sequence corresponding to SEQ ID NO: 417).The HERC2 sequence was detected using a gRNA corresponding to SEQ ID NO:246 (top plot) and the ALDH sequence was detected using a gRNAcorresponding to SEQ ID NO: 425 (bottom plot).

FIG. 81 shows detection of two PNPLA3 SNPs in a target nucleic acidsequence without a native PAM using LbCas12a (SEQ ID NO: 1). Guide RNAscorresponding to SEQ ID NO: 300-SEQ ID NO: 319 were directed to the wildtype (SEQ ID NO: 415, “WT”) sequence on the forward strand at position1-position 20, respectively. gRNAs corresponding to SEQ ID NO: 320-SEQID NO: 339 were directed to the wild type (“WT”) sequence on the reversestrand at position 1-position 20, respectively. gRNAs corresponding toSEQ ID NO: 340-SEQ ID NO: 359 were directed to the mutant (SEQ ID NO:414, “rs738409”) sequence on the forward strand at position 1-position20, respectively. gRNAs corresponding to SEQ ID NO: 360-SEQ ID NO: 379were directed to the mutant (“rs738409”) sequence on the reverse strandat position 1-position 20, respectively. Each gRNA was used to detectfour different target sequences corresponding to the wild type sequence(SEQ ID NO: 415, “WT”), a sequence with a point mutation at a first site(SEQ ID NO: 413, “rs738408”), a sequence with a point mutation at asecond site (SEQ ID NO: 414, “rs738409”), or a sequence with pointmutations at both the first site and the second site (SEQ ID NO: 412,“rs738409+rs738408”).

FIG. 82 shows detection of single point mutations at different positionsalong target RNA sequence (SEQ ID NO: 748,UGGACAAAGCGUCUACGCUGCAGUCCUCGCUCACUGGGCA) using LbuCas13a (SEQ ID NO:104). Data is not shown for wild type positions (black circles labeledwith “WT”). Detection of the wild type sequence is shown in the squaremarked “WT” at SNP position 1. Detection of a negative control (water)is shown in the square marked “None” at position “None.” The targetswere detected using a gRNA corresponding to SEQ ID NO: 507(GGCCACCCCAAAAAUGAAGGGGACUAAAACAAGCGAGGACUGCAGCGUAGA).

FIG. 83 shows detection of single point mutations at different positionsalong target ssDNA (SEQ ID NO: 749,TTTTGGACAAAGCGTCTACGCTGCAGTCCTCGCTCACTGGGCACGGTG) sequence usingLbuCas13a (SEQ ID NO: 104). Data is not shown for wild type positions(black circles labeled with “WT”). Detection of the wild type sequenceis shown in the square marked “WT” at SNP position 1. Detection of anegative control (water) is shown in the square marked “None” atposition “None.” The targets were detected using a gRNA corresponding toSEQ ID NO: 507.

FIG. 84 shows detection of a Chlamydia trachomatis target nucleic acidsequence with LbuCas13a (SEQ ID NO: 104) following polymerase chainreaction (PCR) amplification and in vitro transcription (IVT) of samplesthat were either positive or negative for Chlamydia. Targets weredetected with either a gRNA targeted to Chlamydia 5S rRNA (SEQ ID NO:418), a gRNA targeted to Chlamydia 16S rRNA (SEQ ID NO: 419), or anoff-target gRNA (SEQ ID NO: 420).

FIG. 85 shows heatmaps of the fluorescence detected in FIG. 84 (right).Panels on the right indicate the maximum fluorescent rate detected witheither a gRNA targeting a Chlamydia 16S RNA sequence (SEQ ID NO: 419,“16S gRNA”), a gRNA targeting a Chlamydia 5S RNA sequence (SEQ ID NO:418, “5S gRNA”), or a gRNA not directed to a Chlamydia target sequence(SEQ ID NO: 420, “off-target gRNA”). Shaded boxes in the left column(“Ct”) indicate that the sample was positive for Chlamydia.

FIG. 86 shows trans cleavage rates of different Cas12 variants uponcomplex formation with a gRNA and a target sequence comprising differentPAM sequences. Individual plots show trans cleavage rates for each Cas12variant, and each plot illustrates the cleavage rate for targetsequences comprising different PAM sequences. PAM sequences and thesequences of the target and non-target strands are provided in TABLE 29.Figure discloses “TTTT”, “TTTG”, “TTTC”, “TTTA”, “TTGA”, “TTCA”, “TTAA”,“TGTA”, “TCTA”, “TATA”, “GTTA”, “CTTA”, and “ATTA” as SEQ ID NOS 381-393respectively.

FIG. 87A shows a schematic of a Cas protein, gRNA, and target sequencecomplex comprising either a single base pair mismatch (top) or a doublebase pair mismatch (bottom) between the gRNA and the target sequence.

FIG. 87B shows trans cleavage activity of different Cas12 programmablenuclease variants of SEQ ID NO: 571-SEQ ID NO: 577, SEQ ID NO: 11, SEQID NO: 578-SEQ ID NO: 589, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:590-SEQ ID NO: 598, SEQ ID NO: 580, and SEQ ID NO: 599-SEQ ID NO: 602,and SEQ ID NO: 2 upon complex formation with a gRNA and a targetsequence having either a single base pair mismatch (top) or a doublebase pair mismatch (bottom). Trans cleavage activity was tested formismatches at different positions relative to the PAM sequence. Singleor double mismatches were introduced in the first (“1MM”), fifth(“5MM”), tenth (“10MM”), fifteenth (“15MM”), and twentieth (“20MM”)nucleotide position after the PAM (TTTA (SEQ ID NO: 384)). PAM sequencesand the sequences of the target and non-target strands are provided inTABLE 29.

FIG. 88 shows trans cleavage activity of different Cas12 variants of SEQID NO: 571-SEQ ID NO: 577, SEQ ID NO: 11, SEQ ID NO: 578-SEQ ID NO: 589,SEQ ID NO: 1, and SEQ ID NO: 3 at different concentrations of NaCl.

FIG. 89 shows urea PAGE gels of pre-crRNA processing activity ofdifferent Cas12 variants of SEQ ID NO: 571-SEQ ID NO: 577, SEQ ID NO:11, SEQ ID NO: 578-SEQ ID NO: 589, SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 590-SEQ ID NO: 598, SEQ ID NO: 580, SEQ ID NO: 599-SEQ ID NO: 602,and SEQ ID NO: 2 in the presence (“+”) or absence (“−”) of a Casprotein. Bands shown are RNA bands.

FIG. 90 shows trans cleavage activity of different Cas12 variants of SEQID NO: 571-SEQ ID NO: 577, SEQ ID NO: 11, SEQ ID NO: 578-SEQ ID NO: 589,SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 649-SEQ ID NO: 598, SEQ ID NO:580, SEQ ID NO: 599-SEQ ID NO: 602, and SEQ ID NO: 2 in the presence ofdifferent crRNAs based on the native crRNAs found in the CRISPR locusfor native Cas12 proteins. Pre-crRNA sequences are provided in TABLE 30.The target sequences is set forth in SEQ ID NO: 670.

FIG. 91 shows cis cleavage activity of different Cas12 variants of SEQID NO: 571-SEQ ID NO: 577, SEQ ID NO: 11, SEQ ID NO: 578-SEQ ID NO: 589,SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 590-SEQ ID NO: 598, SEQ ID NO:580, SEQ ID NO: 599-SEQ ID NO: 602, and SEQ ID NO: 2 after incubationwith a target nucleic acid sequence for 10 minutes. Cleavage with BamHIis shown as a cleavage positive control.

FIG. 92 shows sequence alignments of the repeat region of differentCas12 variants aligned to the repeat sequence of LbCas12a (SEQ ID NO:1). Repeat sequences of the Cas12 variants correspond to SEQ ID NO:508-SEQ ID NO: 520 and SEQ ID NO: 522-SEQ ID NO: 536. The repeatsequence of LbCas12a corresponds to SEQ ID NO: 521. Repeat sequences areprovided in TABLE 30.

FIG. 93 shows the results of an assay comparing DETECTR assay efficiencyfor a Cas12 variant of SEQ ID NO: 11 with two different gRNAs. The gRNAcontains either the LbCas12a repeat sequence (“gRNA #1,” SEQ ID NO: 423,UAAUUUCUACUAAGUGUAGAUUCAUCACGCAGCUCAUGCCC) or the Cas12 variant repeatsequence (“gRNA #2,” SEQ ID NO: 424,GUUUGGUACCUUUAUUAAUUUCUACUAAGUGUAGAUUCAUCACGCAGCUCAUGCCC). Figurediscloses SEQ ID NOS 765-766, respectively, in order of appearance.

DETAILED DESCRIPTION

Disclosed herein are compositions, kits and methods related to improvedCas12 and other Cas protein activity. Through compositions and kitsdisclosed herein and practice of methods disclosed herein, one attainsimproved Cas activity such as Cas12 activity relative to Cas proteins inthe art such as LbCas12a. Improved and in some cases high performanceCas12 proteins and conditions are disclosed herein.

The capability to quickly and accurately detect the presence of a targetnucleic acid can provide valuable information associated with thepresence of the target nucleic acid. For example, the capability toquickly and accurately detect the presence of an ailment providesvaluable information and leads to actions to reduce the progression ortransmission of the ailment. Detection of a target nucleic acid moleculeencoding a specific sequence using a programmable nuclease provides amethod for efficiently and accurately detecting the presence of thenucleic acid molecule of interest. There exists a need for highlyefficient, rapid, and accurate reactions for detecting whether a targetnucleic acid is present in a sample. The present disclosure providescompositions and methods for detecting a target nucleic acid in a sampleusing a programmable nuclease in a reaction. The reaction is sometimesreferred to as a DETECTR reaction. The present disclosure providesvarious methods, reagents, enzymes, and kits for rapid tests, which mayquickly assess whether a target nucleic acid is present in a sample byusing a programmable nuclease that can detect the presence of a nucleicacid of interest (e.g., a deoxyribonucleic acid or a deoxyribonucleicacid amplicon of the nucleic acid of interest, which can be the targetdeoxyribonucleic acid) and generating a detectable signal indicating thepresence of said nucleic acid of interest. The methods or reagents maybe used as a point of care diagnostic or as a lab test for detection ofa target nucleic acid and, thereby, detection of a condition in asubject from which the sample was taken. The methods or reagents may beused in various sites or locations, such as in laboratories, inhospitals, in physician offices/laboratories (POLs), in clinics, atremotes sites, or at home. Sometimes, the present disclosure providesvarious devices, systems, fluidic devices, and kits for consumer geneticuse or for over the counter use.

The methods of the present disclosure include providing a programmablenuclease and a guide nucleic acid, wherein the guide nucleic acid isreverse complementary to a target nucleic acid of interest. The targetnucleic acid may be a segment of a nucleic acid sequence of interest.The target nucleic acid may be a gene or a segment of a gene. When theguide nucleic acid hybridizes to the target nucleic acid of interest,the programmable nuclease is activated and exhibits sequence-independentcleavage of a nucleic acid of a reporter. The reporter further comprisesa detection moiety, which is released upon sequence-independent cleavageof the nucleic acid of the reporter, and produces a detectable signal.The detectable signal can be measured and quantified to determine thepresence or absence of the target nucleic acid in the sample and furtherquantify the target nucleic acid when present.

Detecting target nucleic acids in a sample using these methods is highlyunpredictable, as the reaction itself can comprise reagents that inhibitsequence-independent cleavage by an activated programmable nuclease. Forexample, a sample comprising the target nucleic acid may first need tobe lysed. The sample can be further subject to various sample prep stepsincluding filtration, amplification, reverse transcription, and in vitrotranscription. Each of these steps can allow for reagents that mayinhibit an activated programmable nuclease from sequence independentcleavage of the nucleic acid of a reporter, thereby dampening thedetectable signal. As one example, enzymes and/or salts in the buffersfor lysing a sample may inhibit an activated programmable nuclease fromsequence independent cleavage of the nucleic acid of a reporter. Asanother example, salts in the buffer for amplification, reversetranscription, and/or transcription of a target nucleic acid may inhibitan activated programmable nuclease from sequence independent cleavage ofthe nucleic acid of a reporter. As another example, the pH in the bufferof the unlysed sample, the lysis buffer, or the buffer foramplification, reverse transcription, and/or transcription of a targetnucleic acid may inhibit an activated programmable nuclease fromsequence independent cleavage of the nucleic acid of a reporter. In yetanother example, amplification of a target nucleic acid comprises excessprimer and may generate ssDNA that outcompete the nucleic acid of areporter for cleavage by the activated programmable nuclease, therebydampening the detectable signal. The compositions and methods disclosedherein identify volumes of the detection reaction to volumes of thesample, which provide for a strong detectable signal (in the presence ofthe target nucleic acid), thereby alleviating dampened detectablesignals. The compositions and methods disclosed herein also identifyratios of the nucleic acid of the reporter to target and non-targetnucleic acids, which provide for a strong detectable signal (in thepresence of the target nucleic acid), thereby alleviating dampeneddetectable signals.

Also disclosed herein are methods of assaying for a target nucleic acid.The compositions, kits and methods related to improved Cas12 and otherCas protein activity may be implemented in methods of assaying for atarget nucleic acid. In some embodiments, a method of assaying for atarget nucleic acid in a sample, comprises: contacting the sample to acomplex comprising a guide nucleic acid comprising a segment that isreverse complementary to a segment of the target nucleic acid and aprogrammable nuclease that exhibits sequence independent cleavage uponforming a complex comprising the segment of the guide nucleic acidbinding to the segment of the target nucleic acid, wherein the samplecomprises at least one nucleic acid comprising at least 50% sequenceidentity to the segment of the target nucleic acid; and assaying forcleavage of at least one detector nucleic acids (also referred to hereinas “nucleic acid of the reporter”) of a population of detector nucleicacids, wherein the cleavage indicates a presence of the target nucleicacid in the sample and wherein absence of the cleavage indicates anabsence of the target nucleic acid in the sample. The target nucleicacid can be from 0.05% to 20% of total nucleic acids in the sample.Sometimes, the target nucleic acid is from 0.1% to 10% of the totalnucleic acids in the sample. The target nucleic acid, in some cases, isfrom 0.1% to 5% of the total nucleic acids in the sample. Often, asample comprises the segment of the target nucleic acid and at least onenucleic acid comprising less than 100% sequence identity to the segmentof the target nucleic acid but no less than 50% sequence identity to thesegment of the target nucleic acid. For example, the segment of thetarget nucleic acid comprises a mutation as compared to at least onenucleic acid comprising less than 100% sequence identity to the segmentof the target nucleic acid but no less than 50% sequence identity to thesegment of the target nucleic acid. Often, the segment of the targetnucleic acid comprises a single nucleotide mutation as compared to atleast one nucleic acid comprising less than 100% sequence identity tothe segment of the target nucleic acid but no less than 50% sequenceidentity to the segment of the target nucleic acid.

The segment of the target nucleic acid often comprises a singlenucleotide mutation wherein the single nucleotide mutation comprises asingle nucleotide polymorphism (SNP), which is a single base pairvariation in a DNA sequence present in less than 1% of a population.Sometimes, the segment of the target nucleic acid comprises a singlenucleotide mutation, wherein the single nucleotide mutation comprisesthe wild type variant of the SNP. The single nucleotide mutation or SNPcan be associated with a phenotype of the sample or a phenotype of theorganism from which the sample was taken. The SNP, in some cases, isassociated with altered phenotype from wild type phenotype. Often, thesingle nucleotide mutation or SNP is associated with a disease such ascancer or a genetic disorder. The single nucleotide mutation or SNP canbe encoded in the sequence of a target nucleic acid from the germline ofan organism or can be encoded in a target nucleic acid from a diseasedcell, such as a cancer cell. The SNP can be a synonymous substitution ora nonsynonymous substitution. The nonsynonymous substitution can be amissense substitution or a nonsense point mutation. The synonymoussubstitution can be a silent substitution (e.g., a substitution whichdoes not change the amino acid sequence of an encoded protein). Thesegment of the target nucleic acid often comprises a deletion, forexample a deletion of one or more base pairs from an exon sequence. Thedeletion can be associated with a phenotype of the sample or a phenotypeof the organism from which the sample was taken. The deletion, in somecases, is associated with altered phenotype from wild type phenotype.Often, the deletion is associated with a disease such as cancer or agenetic disorder. The deletion can be encoded in the sequence of atarget nucleic acid from the germline of an organism or can be encodedin a target nucleic acid from a diseased cell, such as a cancer cell.The target nucleic acid can be DNA or RNA. Assaying of a target nucleicacid can be used to diagnose or identify diseases associated with targetnucleic acid. The methods described herein use a programmable nuclease,such as the CRISPR/Cas system, to detect a target nucleic acid.

Often, a method disclosed herein comprises: contacting a programmablenuclease comprising a polypeptide having endonuclease activity and aguide nucleic acid to a target nucleic acid in a buffer comprisingheparin. The heparin is present, for example, at a concentration of from1 to 100 μg/ml heparin. Often, the heparin is present at a concentrationof from 40 to 60 μg/ml heparin. Sometimes, the heparin is present at aconcentration 50 μg/ml heparin. Often, the buffer comprises NaCl. TheNaCl is present, for example, at a concentration of from 1 to 200 mMNaCl. Sometimes, the NaCl is present at a concentration of from 80 to120 mM NaCl. Often, the NaCl is present at a concentration of 100 mMNaCl. The target nucleic acid can be a substrate target nucleic acid.Sometimes, the substrate nucleic acid comprises a cancer allele. Often,the cancer allele is present at a low concentration relative to a wildtype allele. Sometimes, the substrate target nucleic acid comprises asplice variant. The substrate target nucleic acid often comprises anedited base. The substrate target nucleic acid sometimes comprises abisulfite-treated base. Often, the substrate target nucleic acidcomprises a segment that is reverse complementary to a segment of theguide nucleic acid.

Assaying of a target nucleic acid comprising a single nucleotidemutation can be difficult, especially in the presence of a nucleic acidcomprising a variant of the single nucleotide mutation because there isonly one nucleotide difference between the sequences of these nucleicacids. Additionally, it is often difficult to assay for the targetnucleic acid comprising the single nucleotide mutation when the samplecomprising the target nucleic acid also comprises more of the nucleicacid comprising the variant of the single nucleotide mutation than thetarget nucleic acid comprising the single nucleotide mutation. Often,the variant is the wild type variant of the single nucleotide mutation.Sometimes, the single nucleotide mutation is the wild variant of a SNP.

The methods described herein can enhance the assay detection a targetnucleic acid. For example, a buffer comprising heparin and NaClincreases the discrimination of a programmable nuclease between thetarget nucleic acid comprising a single nucleotide mutation and othernucleic acids comprising a variant of the single nucleotide mutation.

Amplification methods can also enhance the assay detection of the targetnucleic acid. For example, a PAM target nucleic acid comprising asequence encoding a PAM sequence (e.g., TTTN or dUdUdUN) is produced byamplifying the target nucleic acid segment using a primer having aregion that is reverse complementary to the target nucleic acid segmentand a region that has a PAM sequence reverse complement, therebygenerating a PAM target nucleic acid having a PAM sequence adjacent totarget sequence of an amplification product. Often, the primer is theforward primer comprises the sequence encoding the PAM and has 1-8nucleotides from the 3′ end of the sequence encoding the PAM. Often, thesingle nucleotide mutation in the target nucleic acid sequence is 5-9nucleotides downstream of the 5′ end of the target nucleic acid segmentwherein the target nucleic acid segment is a segment that binds to asegment of the guide nucleic acid that is reverse complementary to itand comprises the sequence encoding the PAM. Additional amplificationstrategies for enhancing the assay detection of the target nucleic acidinclude, but are not limited to, amplification with a blocking primer,wherein the blocking primer binds to variant of the single nucleotidemutation of the target nucleic acid, co-amplification at lowerdenaturation temperature-PCR (COLD-PCR), such as full COLD-PCR and fastCOLD-PCR, allele-specific PCR, targeting the nucleic acids comprising avariant of the single nucleotide mutation with a protein allowing fortheir removal, or targeting the target nucleic acids with a proteinallowing for the removal of the other nucleic acids, or any combinationthereof.

Further disclosed herein are methods of assaying for a target nucleicacid, wherein the target nucleic acid segment lacks a PAM sequence. Forexample, a method of assaying for a target nucleic acid in a samplecomprising: producing a PAM target nucleic acid comprising a sequenceencoding a PAM by amplifying the target nucleic acid of the sample usingprimers comprising the encoding the PAM; contacting the PAM targetnucleic acid to a complex comprising a guide nucleic acid comprising asegment that is reverse complementary to a segment of the PAM targetnucleic acid and a programmable nuclease that exhibits sequenceindependent cleavage upon forming a complex comprising the segment ofthe guide nucleic acid binding to the segment of the PAM target nucleicacid; and assaying for a signal indicating cleavage of at least somedetector nucleic acids of a population of detector nucleic acids,wherein the signal indicates a presence of the target nucleic acid inthe sample and wherein the absence of the signal indicates an absence ofthe target nucleic acid in the sample. Sometimes, a method of assayingfor a target nucleic acid segment in a sample, wherein the targetnucleic acid segment lacks a PAM sequence, comprises amplifying thetarget nucleic acid segment using a primer having a region that isreverse complementary to the target nucleic acid segment and a regionthat has a PAM sequence reverse complement, thereby generating a PAMtarget nucleic acid having a PAM sequence adjacent to target sequence ofan amplification product; contacting the PAM target nucleic acid toPAM-dependent sequence specific nuclease complex comprising a guidenucleic acid and a programmable nuclease that exhibits sequenceindependent cleavage upon forming a complex comprising the segment ofthe guide nucleic acid binding to the segment of the PAM target nucleicacid; and assaying for cleavage of at least one detector nucleic acid ofa population of detector nucleic acids, wherein the cleavage indicates apresence of the target nucleic acid in the sample and wherein theabsence of the cleavage indicates an absence of the target nucleic acidin the sample. The sequence encoding the PAM can comprise TTTN.Sometimes, the sequence encoding the PAM comprises dUdUdUN. Often, aforward primer of the primers comprises the sequence encoding the PAMand has one to ten nucleotides from the 3′ end of the sequence encodingthe PAM. These nucleotides can be referred to as extension nucleotides.In some embodiments, extensions with 10 nucleotides or fewer may producespecific detection with the guide RNA. In some embodiments, extensionswith greater than 12 nucleotides may self-activate, resulting in reduceddetection specificity for the target sequence. Extensions between 5nucleotides and 10 nucleotides may provide sufficient overlap with thetarget sequence to anneal to the target sequence with an annealingtemperature amenable to detection. In some embodiments, an extension maycomprise 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 3′ end of thesequence encoding the PAM. Sometimes, a mismatch for single nucleotidepolymorphism (SNP) detection is 3-10 nucleotides downstream of the PAMin PAM target nucleic acid. This allows for detection of any targetnucleic acid by a programmable nuclease. The methods described hereinuse a programmable nuclease, such as the CRISPR/Cas system, to detect atarget nucleic acid. Often, the programmable nuclease is Cas12.

A target nucleic acid is required to have a PAM sequence for binding andtrans cleavage activation of some programmable nucleases complexed witha guide nucleic acid. However, there are many target nucleic acids ofinterest that do not encode for the PAM sequence. Therefore, there is aneed for strategies to allow for binding and trans cleavage activationof the programmable nucleases complexed with a guide nucleic acid usingany target nucleic sequence of interest.

The methods describe herein use amplification techniques to insert a PAMsequence into the target nucleic acid for recognition by theprogrammable nuclease complexed with the guide nucleic acid.

Sample

A number of samples are consistent with the compositions and methodsdisclosed herein. The samples, as described herein, are compatible withthe DETECTR assay methods disclosed herein. The samples, as describedherein, are compatible with any of the programmable nucleases disclosedherein (e.g., a programmable nuclease with at least 60% sequenceidentity to SEQ ID NO: 11) and use of said programmable nuclease in amethod of detecting a target nucleic acid. The samples, as describedherein, are compatible with any of the compositions comprising aprogrammable nuclease and a buffer, which has been developed to improvethe function of the programmable nuclease (e.g., a programmable nucleaseand a buffer with low salt (about 110 mM or less) and a pH of 7 to 8)and use of said compositions in a method of detecting a target nucleicacid. The samples, as described herein, are compatible with any of themethods disclosed herein including methods of assaying for at least onebase difference (e.g., assaying for a SNP or a base mutation) in atarget nucleic acid sequence, methods of assaying for a target nucleicacid that lacks a PAM by amplifying the target nucleic acid sequence tointroduce a PAM, and compositions used in introducing a PAM viaamplification into the target nucleic acid sequence. Described hereinare sample that contain deoxyribonucleic acid (DNA), ribonucleic acid(RNA), or both, which can be detected using a programmable nuclease,such as a Type V CRISPR/Cas enzyme (e.g., a Cas12 such as Cas12 is aCas12a, Cas12b, Cas12c, Cas12d (also referred to as CasY), or Cas12e ora Cas14 such as Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g,or Cas14h) or a Type VI CRISPR enzyme (e.g., a Cas13 such as Cas13a,Cas13b, Cas13c, Cas13d, or Cas13e). As described herein, programmablenucleases are activated upon binding to a target nucleic acid ofinterest in a sample upon hybridization of a guide nucleic acid to thetarget nucleic acid. Subsequently, the activated programmable nucleasesexhibit sequence-independent cleavage of a nucleic acid in a reporter.The reporter additionally includes a detectable moiety, which isreleased upon sequence-independent cleavage of the nucleic acid in thereporter. The detectable moiety emits a detectable signal, which can bemeasured by various methods (e.g., spectrophotometry, fluorescencemeasurements, electrochemical measurements).

Various sample types comprising a target nucleic acid of interest areconsistent with the present disclosure. These samples can comprise atarget nucleic acid sequence for detection. In some embodiments, thedetection of the target nucleic indicates an ailment, such as a disease,cancer, or genetic disorder, or genetic information, such as forphenotyping, genotyping, or determining ancestry and are compatible withthe reagents and support mediums as described herein. Generally, asample from an individual or an animal or an environmental sample can beobtained to test for presence of a disease, cancer, genetic disorder, orany mutation of interest. A biological sample from the individual may beblood, serum, plasma, saliva, urine, mucosal sample, peritoneal sample,cerebrospinal fluid, gastric secretions, nasal secretions, sputum,pharyngeal exudates, urethral or vaginal secretions, an exudate, aneffusion, or tissue. A tissue sample may be dissociated or liquifiedprior to application to detection system of the present disclosure. Asample from an environment may be from soil, air, or water. In someinstances, the environmental sample is taken as a swab from a surface ofinterest or taken directly from the surface of interest. In someinstances, the raw sample is applied to the detection system. In someinstances, the sample is diluted with a buffer or a fluid orconcentrated prior to application to the detection system or be appliedneat to the detection system. Sometimes, the sample is contained in nomore 20 μl. The sample, in some cases, is contained in no more than 1,5, 10, 15, 20, 25, 30, 35 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100,200, 300, 400, 500 μl, or any of value from 1 μl to 500 μl, preferablyfrom 10 μL to 200 μL, or more preferably from 50 μL to 100 μL.Sometimes, the sample is contained in more than 500 μl.

In some embodiments, the target nucleic acid is single-stranded DNA. Themethods, reagents, enzymes, and kits disclosed herein may enable thedirect detection of a DNA encoding a sequence of interest, in particulara single-stranded DNA encoding a sequence of interest, withouttranscribing the DNA into RNA, for example, by using an RNA polymerase.The compositions and methods disclosed herein may enable the detectionof target nucleic acid that is an amplified nucleic acid of a nucleicacid of interest. In some embodiments, the target nucleic acid is acDNA, genomic DNA, an amplicon of genomic DNA or a DNA amplicon of anRNA. A nucleic acid can encode a sequence from a genomic locus. In somecases, the target nucleic acid that binds to the guide nucleic acid isfrom 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides in length. Thenucleic acid can be from 10 to 90, from 20 to 80, from 30 to 70, or from40 to 60 nucleotides in length. A nucleic acid can be 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90,or 100 nucleotides in length. The target nucleic acid can encode asequence reverse complementary to a guide nucleic acid sequence.

In some instances, the sample is taken from single-cell eukaryoticorganisms; a plant or a plant cell; an algal cell; a fungal cell; ananimal cell, tissue, or organ; a cell, tissue, or organ from aninvertebrate animal; a cell, tissue, fluid, or organ from a vertebrateanimal such as fish, amphibian, reptile, bird, and mammal; a cell,tissue, fluid, or organ from a mammal such as a human, a non-humanprimate, an ungulate, a feline, a bovine, an ovine, and a caprine. Insome instances, the sample is taken from nematodes, protozoans,helminths, or malarial parasites. In some cases, the sample comprisesnucleic acids from a cell lysate from a eukaryotic cell, a mammaliancell, a human cell, a prokaryotic cell, or a plant cell. In some cases,the sample comprises nucleic acids expressed from a cell.

The sample described herein may comprise at least one target nucleicacid. The target nucleic acid comprises a segment that is reversecomplementary to a segment of a guide nucleic acid. Often, the samplecomprises the segment of the target nucleic acid and at least onenucleic acid comprising at least 50% sequence identity to a segment ofthe target nucleic acid. Sometimes, the at least one nucleic acidcomprises a segment comprising at least 60%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to thesegment of the target nucleic acid. Often, a sample comprises thesegment of the target nucleic acid and at least one nucleic acid asegment comprising less than 100% sequence identity to the targetnucleic acid but no less than 50% sequence identity to the segment ofthe target nucleic acid. Sometimes, a sample comprises the segment ofthe target nucleic acid and at least one nucleic acid a segmentcomprising less than 100% sequence identity to the target nucleic acidbut no less than 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to the segment of the targetnucleic acid. For example, the segment of the target nucleic acidcomprises a mutation as compared to at least one nucleic acid comprisinga segment comprising less than 100% sequence identity to the segment ofthe target nucleic acid but no less than 50% sequence identity to thesegment of the target nucleic acid. Sometimes, the segment of the targetnucleic acid comprises a mutation as compared to at least one nucleicacid comprising a segment comprising less than 100% sequence identity tothe segment of the target nucleic acid but no less than 60%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the segment of the target nucleic acid. Often, the segmentof the target nucleic acid comprises a mutation as compared to at leastone nucleic acid comprising a segment comprising less than 100% sequenceidentity to the segment of the target nucleic acid but no less than 50%sequence identity to the segment of the target nucleic acid. Themutation can be a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or more nucleotides. Often, the mutation is asingle nucleotide mutation. The single nucleotide mutation can be asingle nucleotide polymorphism (SNP), which is a single base pairvariation in a DNA sequence present in less than 1% of a population.Sometimes, the target nucleic acid comprises a single nucleotidemutation, wherein the single nucleotide mutation comprises the wild typevariant of the SNP. The single nucleotide mutation or SNP can beassociated with a phenotype of the sample or a phenotype of the organismfrom which the sample was taken. The SNP, in some cases, is associatedwith altered phenotype from wild type phenotype. Often, the segment ofthe target nucleic acid sequence comprises a deletion as compared to atleast one nucleic acid comprising a segment comprising less than 100%sequence identity to the segment of the target nucleic acid but no lessthan 50% sequence identity to the segment of the target nucleic acid.The mutation can be a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. The mutation can bea deletion of about 5, about 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45, about 50, about 55, about 60, about 65,about 70, about 75, about 80, about 85, about 90, about 95, about 100,about 200, about 300, about 400, about 500, about 600, about 700, about800, about 900, or about 1000 nucleotides. The mutation can be adeletion of from 1 to 5, from 5 to 10, from 10 to 15, from 15 to 20,from 20 to 25, from 25 to 30, from 30 to 35, from 35 to 40, from 40 to45, from 45 to 50, from 50 to 55, from 55 to 60, from 60 to 65, from 65to 70, from 70 to 75, from 75 to 80, from 80 to 85, from 85 to 90, from90 to 95, from 95 to 100, from 100 to 200, from 200 to 300, from 300 to400, from 400 to 500, from 500 to 600, from 600 to 700, from 700 to 800,from 800 to 900, from 900 to 1000, from 1 to 50, from 1 to 100, from 25to 50, from 25 to 100, from 50 to 100, from 100 to 500, from 100 to1000, or from 500 to 1000 nucleotides. The segment of the target nucleicacid that the guide nucleic acid of the methods describe herein binds tocomprises the mutation, such as the SNP or the deletion. The mutationcan be a single nucleotide mutation or a SNP. The SNP can be asynonymous substitution or a nonsynonymous substitution. Thenonsynonymous substitution can be a missense substitution or a nonsensepoint mutation. The synonymous substitution can be a silentsubstitution. The mutation can be a deletion of one or more nucleotides.Often, the single nucleotide mutation, SNP, or deletion is associatedwith a disease such as cancer or a genetic disorder. The mutation, suchas a single nucleotide mutation, a SNP, or a deletion, can be encoded inthe sequence of a target nucleic acid from the germline of an organismor can be encoded in a target nucleic acid from a diseased cell, such asa cancer cell.

The sample used for disease testing may comprise at least one targetnucleic acid that can bind to a guide nucleic acid of the reagentsdescribed herein. The sample used for disease testing may comprise atleast nucleic acid of interest that is amplified to produce a targetnucleic acid that can bind to a guide nucleic acid of the reagentsdescribed herein. The nucleic acid of interest can comprise DNA, RNA, ora combination thereof.

The target nucleic acid (e.g., a target DNA) may be a portion of anucleic acid from a virus or a bacterium or other agents responsible fora disease in the sample. The target nucleic acid may be a portion of anucleic acid from a gene expressed in a cancer or genetic disorder inthe sample. In some cases, the sequence is a segment of a target nucleicacid sequence. A segment of a target nucleic acid sequence can be from agenomic locus, a transcribed mRNA, or a reverse transcribed cDNA. Asegment of a target nucleic acid sequence can be from 5 to 100, 5 to 90,5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20,5 to 15, or 5 to 10 nucleotides in length. A segment of a target nucleicacid sequence can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides in length.The sequence of the target nucleic acid segment can be reversecomplementary to a segment of a guide nucleic acid sequence. The targetnucleic acid may comprise a genetic variation (e.g., a single nucleotidepolymorphism), with respect to a standard sample, associated with adisease phenotype or disease predisposition. The target nucleic acid maybe an amplicon of a portion of an RNA, may be a DNA, or may be a DNAamplicon from any organism in the sample.

In some embodiments, the target nucleic acid sequence comprises anucleic acid sequence of a virus or a bacterium or other agentsresponsible for a disease in the sample. In some embodiments, the targetnucleic acid comprises DNA that is reverse transcribed from RNA using areverse transcriptase prior to detection by a programmable nucleaseusing the compositions, systems, and methods disclosed herein. Thetarget nucleic acid, in some cases, is a portion of a nucleic acid froma sexually transmitted infection or a contagious disease, in the sample.In some cases, the target nucleic acid is a portion of a nucleic acidfrom a genomic locus, or any DNA amplicon, such as a reverse transcribedmRNA or a cDNA from a gene locus, a transcribed mRNA, or a reversetranscribed cDNA from a gene locus in at least one of: humanimmunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia,gonorrhea, syphilis, trichomoniasis, sexually transmitted infection,malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis. Pathogensinclude viruses, fungi, helminths, protozoa, malarial parasites,Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites.Helminths include roundworms, heartworms, and phytophagous nematodes,flukes, Acanthocephala, and tapeworms. Protozoan infections includeinfections from Giardia spp., Trichomonas spp., African trypanosomiasis,amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease,coccidiosis, malaria and toxoplasmosis. Examples of pathogens such asparasitic/protozoan pathogens include, but are not limited to:Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasmagondii. Fungal pathogens include, but are not limited to Cryptococcusneoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomycesdermatitidis, Chlamydia trachomatis, and Candida albicans. Pathogenicviruses include but are not limited to immunodeficiency virus (e.g.,HIV); influenza virus; dengue; West Nile virus; herpes virus; yellowfever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B;papillomavirus; and the like. Pathogens include, e.g., HIV virus,Mycobacterium tuberculosis, Streptococcus agalactiae,methicillin-resistant Staphylococcus aureus, Legionella pneumophila,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans,Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum,Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae,Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpessimplex virus I, herpes simplex virus II, human serum parvo-like virus,respiratory syncytial virus (RSV), M. genitalium, T. vaginalis,varicella-zoster virus, hepatitis B virus, hepatitis C virus, measlesvirus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus,murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbisvirus, lymphocytic choriomeningitis virus, wart virus, blue tonguevirus, Sendai virus, feline leukemia virus, Reovirus, polio virus,simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus,West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasmagondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense,Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesiabovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica,Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva,Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcusgranulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis,M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and M.pneumoniae. In some cases, the target sequence is a portion of a nucleicacid from a genomic locus, a transcribed mRNA, or a reverse transcribedcDNA from a gene locus of bacterium or other agents responsible for adisease in the sample comprising a mutation that confers resistance to atreatment, such as a single nucleotide mutation that confers resistanceto antibiotic treatment. In some cases, the mutation that confersresistance to a treatment is a deletion.

The sample used for cancer testing may comprise at least one targetnucleic acid that can bind to a guide nucleic acid of the reagentsdescribed herein. The target nucleic acid, in some cases, comprises aportion of a gene comprising a mutation associated with cancer, a genewhose overexpression is associated with cancer, a tumor suppressor gene,an oncogene, a checkpoint inhibitor gene, a gene associated withcellular growth, a gene associated with cellular metabolism, or a geneassociated with cell cycle. Sometimes, the target nucleic acid encodes acancer biomarker, such as a prostate cancer biomarker or non-small celllung cancer. In some cases, the assay can be used to detect “hotspots”in target nucleic acids that can be predictive of lung cancer. In somecases, the target nucleic acid comprises a portion of a nucleic acidthat is associated with a blood fever. In some cases, the target nucleicacid is a portion of a nucleic acid from a genomic locus, any DNAamplicon of, a reverse transcribed mRNA, or a cDNA from a locus of atleast one of: ALK, APC, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1,BRCA2, BRIP1, CASR, CDC73, CDH1, CDK4, CDKN1B, CDKN1C, CDKN2A, CEBPA,CHEK2, CTNNA1, DICERI, DIS3L2, EGFR, EPCAM, FH, FLCN, GATA2, GPC3,GREM1, HOXB13, HRAS, KIT, MAX, MEN1, MET, MITF, MLH1, MSH2, MSH3, MSH6,MUTYH, NBN, NF1, NF2, NTHL1, PALB2, PDGFRA, PHOX2B, PMS2, POLD1, POLE,POT1, PRKAR1A, PTCH1, PTEN, RAD50, RAD51C, RAD51D, RB1, RECQL4, RET,RUNX1, SDHA, SDHAF2, SDHB, SDHC, SDHD, SMAD4, SMARCA4, SMARCB1, SMARCE1,STK11, SUFU, TERC, TERT, TMEM127, TP53, TSC1, TSC2, VHL, WRN, and WT1.Any region of the aforementioned gene loci can be probed for a mutationor deletion using the compositions and methods disclosed herein. Forexample, in the EGFR gene locus, the compositions and methods fordetection disclosed herein can be used to detect a single nucleotidepolymorphism or a deletion. The SNP or deletion can occur in anon-coding region or a coding region. The SNP or deletion can occur inan Exon, such as Exon19.

The sample used for genetic disorder testing may comprise at least onetarget nucleic acid that can bind to a guide nucleic acid of thereagents described herein. In some embodiments, the genetic disorder ishemophilia, sickle cell anemia, 0-thalassemia, Duchene musculardystrophy, severe combined immunodeficiency, Huntington's disease, orcystic fibrosis. The target nucleic acid, in some cases, is from a genewith a mutation associated with a genetic disorder, from a gene whoseoverexpression is associated with a genetic disorder, from a geneassociated with abnormal cellular growth resulting in a geneticdisorder, or from a gene associated with abnormal cellular metabolismresulting in a genetic disorder. In some cases, the target nucleic acidis a nucleic acid from a genomic locus, a transcribed mRNA, or a reversetranscribed mRNA, a DNA amplicon of or a cDNA from a locus of at leastone of: CFTR, FMR1, SMN1, ABCB11, ABCC8, ABCD1, ACAD9, ACADM, ACADVL,ACAT1, ACOX1, ACSF3, ADA, ADAMTS2, ADGRG1, AGA, AGL, AGPS, AGXT, AIRE,ALDH3A2, ALDOB, ALG6, ALMS1, ALPL, AMT, AQP2, ARG1, ARSA, ARSB, ASL,ASNS, ASPA, ASS1, ATM, ATP6V1B1, ATP7A, ATP7B, ATRX, BBS1, BBS10, BBS12,BBS2, BCKDHA, BCKDHB, BCS1L, BLM, BSND, CAPN3, CBS, CDH23, CEP290,CERKL, CHM, CHRNE, CIITA, CLN3, CLN5, CLN6, CLN8, CLRN1, CNGB3, COL27A1,COL4A3, COL4A4, COL4A5, COL7A1, CPS1, CPT1A, CPT2, CRB1, CTNS, CTSK,CYBA, CYBB, CYP1IB1, CYP11B2, CYP17A1, CYP19A1, CYP27A1, DBT, DCLRElC,DHCR7, DHDDS, DLD, DMD, DNAH5, DNAI1, DNAI2, DYSF, EDA, EIF2B5, EMD,ERCC6, ERCC8, ESCO2, ETFA, ETFDH, ETHE1, EVC, EVC2, EYS, F9, FAH,FAM161A, FANCA, FANCC, FANCG, FH, FKRP, FKTN, G6PC, GAA, GALC, GALK1,GALT, GAMT, GBA, GBE1, GCDH, GFM1, GJB1, GJB2, GLA, GLB1, GLDC, GLE1,GNE, GNPTAB, GNPTG, GNS, GRHPR, HADHA, HAX1, HBA1, HBA2, HBB, HEXA,HEXB, HGSNAT, HLCS, HMGCL, HOGA1, HPS1, HPS3, HSD17B4, HSD3B2, HYAL1,HYLS1, IDS, IDUA, IKBKAP, IL2RG, IVD, KCNJ11, LAMA2, LAMA3, LAMB3,LAMC2, LCA5, LDLR, LDLRAP1, LHX3, LIFR, LIPA, LOXHD1, LPL, LRPPRC,MAN2B1, MCOLN1, MED17, MESP2, MFSD8, MKS1, MLC1, MMAA, MMAB, MMACHC,MMADHC, MPI, MPL, MPV17, MTHFR, MTM1, MTRR, MTTP, MUT, MYO7A, NAGLU,NAGS, NBN, NDRG1, NDUFAF5, NDUFS6, NEB, NPC1, NPC2, NPHS1, NPHS2, NR2E3,NTRK1, OAT, OPA3, OTC, PAH, PC, PCCA, PCCB, PCDH15, PDHA1, PDHB, PEX1,PEX10, PEX12, PEX2, PEX6, PEX7, PFKM, PHGDH, PKHD1, PMM2, POMGNT1, PPT1,PROP1, PRPS1, PSAP, PTS, PUS1, PYGM, RAB23, RAG2, RAPSN, RARS2, RDH12,RMRP, RPE65, RPGRIP1L, RS1, RTEL1, SACS, SAMHD1, SEPSECS, SGCA, SGCB,SGCG, SGSH, SLC12A3, SLC12A6, SLC17A5, SLC22A5, SLC25A13, SLC25A15,SLC26A2, SLC26A4, SLC35A3, SLC37A4, SLC39A4, SLC4A11, SLC6A8, SLC7A7,SMARCAL1, SMPD1, STAR, SUMF1, TAT, TCIRG1, TECPR2, TFR2, TGM1, TH,TMEM216, TPP1, TRMU, TSFM, TTPA, TYMP, USH1C, USH2A, VPS13A, VPS13B,VPS45, VRK1, VSX2, WNT10A, XPA, XPC, and ZFYVE26.

The sample used for phenotyping testing may comprise at least one targetnucleic acid that can bind to a guide nucleic acid of the reagentsdescribed herein. The target nucleic acid, in some cases, is a nucleicacid encoding a sequence associated with a phenotypic trait.

The sample used for genotyping testing may comprise at least one targetnucleic acid that can bind to a guide nucleic acid of the reagentsdescribed herein. The target nucleic acid, in some cases, is a nucleicacid encoding a sequence associated with a genotype of interest.

The sample used for ancestral testing may comprise at least one targetnucleic acid that can bind to a guide nucleic acid of the reagentsdescribed herein. The target nucleic acid, in some cases, is a nucleicacid encoding a sequence associated with a geographic region of originor ethnic group.

The sample can be used for identifying a disease status. For example, asample is any sample described herein, and is obtained from a subjectfor use in identifying a disease status of a subject. The disease can bea cancer or genetic disorder. Sometimes, a method comprises obtaining aserum sample from a subject; and identifying a disease status of thesubject. Often, the disease status is prostate disease status.

In some instances, the target nucleic acid is a single stranded nucleicacid. Alternatively or in combination, the target nucleic acid is adouble stranded nucleic acid and is prepared into single strandednucleic acids before or upon contacting the reagents. The target nucleicacid may be a reverse transcribed RNA, DNA, DNA amplicon, syntheticnucleic acids, or nucleic acids found in biological or environmentalsamples. The target nucleic acids include but are not limited to mRNA,rRNA, tRNA, non-coding RNA, long non-coding RNA, and microRNA (miRNA).In some cases, the target nucleic acid is single-stranded DNA (ssDNA) ormRNA. In some cases, the target nucleic acid is from a virus, aparasite, or a bacterium described herein. In some cases, the targetnucleic acid is transcribed from a gene as described herein and thenreverse transcribed into a DNA amplicon.

A number of target nucleic acids are consistent with the methods andcompositions disclosed herein. Some methods described herein can detecta target nucleic acid present in the sample in various concentrations oramounts as a target nucleic acid population. In some cases, the samplehas at least 2 target nucleic acids. In some cases, the sample has atleast 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000target nucleic acids. In some cases, the sample as from 1 to 10,000,from 100 to 8000, from 400 to 6000, from 500 to 5000, from 1000 to 4000,or from 2000 to 3000 target nucleic acids. In some cases, the methoddetects target nucleic acid present at least at one copy per 10non-target nucleic acids, 10² non-target nucleic acids, 10³ non-targetnucleic acids, 10⁴ non-target nucleic acids, 10⁵ non-target nucleicacids, 10⁶ non-target nucleic acids, 10⁷ non-target nucleic acids, 10⁸non-target nucleic acids, 10⁹ non-target nucleic acids, or 10¹⁰non-target nucleic acids. Often, the target nucleic acid can be from0.05% to 20% of total nucleic acids in the sample. Sometimes, the targetnucleic acid is from 0.1% to 10% of the total nucleic acids in thesample. The target nucleic acid, in some cases, is from 0.1% to 5% ofthe total nucleic acids in the sample. The target nucleic acid can alsobe from 0.1% to 1% of the total nucleic acids in the sample. The targetnucleic acid can be DNA or RNA. The target nucleic acid can be anyamount less than 100% of the total nucleic acids in the sample. Thetarget nucleic acid can be 100% of the total nucleic acids in thesample.

In some embodiments, the sample comprises a target nucleic acid at aconcentration of less than 1 nM, less than 2 nM, less than 3 nM, lessthan 4 nM, less than 5 nM, less than 6 nM, less than 7 nM, less than 8nM, less than 9 nM, less than 10 nM, less than 20 nM, less than 30 nM,less than 40 nM, less than 50 nM, less than 60 nM, less than 70 nM, lessthan 80 nM, less than 90 nM, less than 100 nM, less than 200 nM, lessthan 300 nM, less than 400 nM, less than 500 nM, less than 600 nM, lessthan 700 nM, less than 800 nM, less than 900 nM, less than 1 μM, lessthan 2 μM, less than 3 μM, less than 4 μM, less than 5 μM, less than 6μM, less than 7 μM, less than 8 μM, less than 9 μM, less than 10 μM,less than 100 μM, or less than 1 mM. In some embodiments, the samplecomprises a target nucleic acid sequence at a concentration of from 1 nMto 2 nM, from 2 nM to 3 nM, from 3 nM to 4 nM, from 4 nM to 5 nM, from 5nM to 6 nM, from 6 nM to 7 nM, from 7 nM to 8 nM, from 8 nM to 9 nM,from 9 nM to 10 nM, from 10 nM to 20 nM, from 20 nM to 30 nM, from 30 nMto 40 nM, from 40 nM to 50 nM, from 50 nM to 60 nM, from 60 nM to 70 nM,from 70 nM to 80 nM, from 80 nM to 90 nM, from 90 nM to 100 nM, from 100nM to 200 nM, from 200 nM to 300 nM, from 300 nM to 400 nM, from 400 nMto 500 nM, from 500 nM to 600 nM, from 600 nM to 700 nM, from 700 nM to800 nM, from 800 nM to 900 nM, from 900 nM to 1 μM, from 1 μM to 2 μM,from 2 μM to 3 μM, from 3 μM to 4 μM, from 4 μM to 5 μM, from 5 μM to 6μM, from 6 μM to 7 μM, from 7 μM to 8 μM, from 8 μM to 9 μM, from 9 μMto 10 μM, from 10 μM to 100 μM, from 100 μM to 1 mM, from 1 nM to 10 nM,from 1 nM to 100 nM, from 1 nM to 1 μM, from 1 nM to 10 μM, from 1 nM to100 μM, from 1 nM to 1 mM, from 10 nM to 100 nM, from 10 nM to 1 μM,from 10 nM to 10 μM, from 10 nM to 100 μM, from 10 nM to 1 mM, from 100nM to 1 μM, from 100 nM to 10 μM, from 100 nM to 100 μM, from 100 nM to1 mM, from 1 μM to 10 μM, from 1 μM to 100 μM, from 1 μM to 1 mM, from10 μM to 100 μM, from 10 μM to 1 mM, or from 100 μM to 1 mM. In someembodiments, the sample comprises a target nucleic acid at aconcentration of from 20 nM to 200 μM, from 50 nM to 100 μM, from 200 nMto 50 μM, from 500 nM to 20 μM, or from 2 μM to 10 μM. In someembodiments, the target nucleic acid is not present in the sample.

In some embodiments, the sample comprises fewer than 10 copies, fewerthan 100 copies, fewer than 1000 copies, fewer than 10,000 copies, fewerthan 100,000 copies, or fewer than 1,000,000 copies of a target nucleicacid sequence. In some embodiments, the sample comprises from 10 copiesto 100 copies, from 100 copies to 1000 copies, from 1000 copies to10,000 copies, from 10,000 copies to 100,000 copies, from 100,000 copiesto 1,000,000 copies, from 10 copies to 1000 copies, from 10 copies to10,000 copies, from 10 copies to 100,000 copies, from 10 copies to1,000,000 copies, from 100 copies to 10,000 copies, from 100 copies to100,000 copies, from 100 copies to 1,000,000 copies, from 1,000 copiesto 100,000 copies, or from 1,000 copies to 1,000,000 copies of a targetnucleic acid sequence. In some embodiments, the sample comprises from 10copies to 500,000 copies, from 200 copies to 200,000 copies, from 500copies to 100,000 copies, from 1000 copies to 50,000 copies, from 2000copies to 20,000 copies, from 3000 copies to 10,000 copies, or from 4000copies to 8000 copies. In some embodiments, the target nucleic acid isnot present in the sample.

A number of target nucleic acid populations are consistent with themethods and compositions disclosed herein. Some methods described hereincan detect two or more target nucleic acid populations present in thesample in various concentrations or amounts. In some cases, the samplehas at least 2 target nucleic acid populations. In some cases, thesample has at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 targetnucleic acid populations. In some cases, the sample has from 3 to 50,from 5 to 40, or from 10 to 25 target nucleic acid populations. In somecases, the method detects target nucleic acid populations that arepresent at least at one copy per 10¹ non-target nucleic acids, 10²non-target nucleic acids, 10³ non-target nucleic acids, 10⁴ non-targetnucleic acids, 10⁵ non-target nucleic acids, 10⁶ non-target nucleicacids, 10⁷ non-target nucleic acids, 10⁸ non-target nucleic acids, 10⁹non-target nucleic acids, or 10¹⁰ non-target nucleic acids. The targetnucleic acid populations can be present at different concentrations oramounts in the sample.

Additionally, target nucleic acid can be an amplified nucleic acid ofinterest, which can bind to the guide nucleic acid of a programmablenuclease, such as a DNA-activated programmable RNA nuclease. The nucleicacid of interest may be any nucleic acid disclosed herein or from anysample as disclosed herein. This amplification can be thermalamplification (e.g., using PCR) or isothermal amplification. Thisnucleic acid amplification of the sample can improve at least one ofsensitivity, specificity, or accuracy of the detection the targetnucleic acid. The reagents for nucleic acid amplification can comprise arecombinase, a oligonucleotide primer, a single-stranded DNA binding(SSB) protein, and a polymerase. The nucleic acid amplification can betranscription mediated amplification (TMA). Nucleic acid amplificationcan be helicase dependent amplification (HDA) or circular helicasedependent amplification (cHDA). In additional cases, nucleic acidamplification is strand displacement amplification (SDA). The nucleicacid amplification can be recombinase polymerase amplification (RPA).The nucleic acid amplification can be at least one of loop mediatedamplification (LAMP) or the exponential amplification reaction (EXPAR).Nucleic acid amplification is, in some cases, by rolling circleamplification (RCA), ligase chain reaction (LCR), simple methodamplifying RNA targets (SMART), single primer isothermal amplification(SPIA), multiple displacement amplification (MDA), nucleic acid sequencebased amplification (NASBA), hinge-initiated primer-dependentamplification of nucleic acids (HIP), nicking enzyme amplificationreaction (NEAR), or improved multiple displacement amplification (IMDA).The nucleic acid amplification can be performed for no greater than 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 40, 50, or 60 minutes. Sometimes, the nucleic acid amplificationreaction is performed at a temperature of around 20-45° C. The nucleicacid amplification reaction can be performed at a temperature no greaterthan 20° C., 25° C., 30° C., 35° C., 37° C., 40° C., 45° C. The nucleicacid amplification reaction can be performed at a temperature of atleast 20° C., 25° C., 30° C., 35° C., 37° C., 40° C., or 45° C.

In some embodiments, the target nucleic acid as disclosed herein canactivate the programmable nuclease to initiate sequence-independentcleavage of a nucleic acid-based reporter (e.g., a reporter comprising aDNA sequence, a reporter comprising an RNA sequence, or a reportercomprising DNA and RNA). For example, a programmable nuclease of thepresent disclosure is activated by a target DNA to cleave reportershaving an RNA (also referred to herein as an “RNA reporter”).Alternatively, a programmable nuclease of the present disclosure isactivated by a target RNA to cleave reporters having an RNA.Alternatively, a programmable nuclease of the present disclosure isactivated by a target DNA to cleave reporters having a DNA (alsoreferred to herein as a “DNA reporter”). The RNA reporter can comprise asingle-stranded RNA labelled with a detection moiety or can be any RNAreporter as disclosed herein. The DNA reporter can comprise asingle-stranded DNA labelled with a detection moiety or can be any DNAreporter as disclosed herein.

In some embodiments, the target nucleic acid as described in the methodsherein does not initially comprise a PAM sequence. However, any targetnucleic acid of interest may be generated using the methods describedherein to comprise a PAM sequence, and thus be a PAM target nucleicacid. A PAM target nucleic acid, as used herein, refers to a targetnucleic acid that has been amplified to insert a PAM sequence that isrecognized by a CRISPR/Cas system.

Any of the above disclosed samples are consistent with the methods,compositions, reagents, enzymes, and kits disclosed herein and can beused as a companion diagnostic with any of the diseases disclosedherein, or can be used in reagent kits, point-of-care diagnostics, orover-the-counter diagnostics.

Reagents for Detection of Target Nucleic Acids

Disclosed herein are methods of assaying for a target nucleic acid asdescribed herein. The reagents for detection of target nucleic acids, asdescribed herein, are compatible with the DETECTR assay methodsdisclosed herein. The reagents for detection of target nucleic acids, asdescribed herein, are compatible with any of the programmable nucleasesdisclosed herein (e.g., a programmable nuclease with at least 60%sequence identity to SEQ ID NO: 11) and use of said programmablenuclease in a method of detecting a target nucleic acid. The reagentsfor detection of target nucleic acids, as described herein, arecompatible with any of the compositions comprising a programmablenuclease and a buffer, which has been developed to improve the functionof the programmable nuclease (e.g., a programmable nuclease and a bufferwith low salt (about 110 mM or less) and a pH of 7 to 8) and use of saidcompositions in a method of detecting a target nucleic acid. Thereagents for detection of target nucleic acids, as described herein, arecompatible with any of the methods disclosed herein including methods ofassaying for at least one base difference (e.g., assaying for a SNP or abase mutation) in a target nucleic acid sequence, methods of assayingfor a target nucleic acid that lacks a PAM by amplifying the targetnucleic acid sequence to introduce a PAM, and compositions used inintroducing a PAM via amplification into the target nucleic acidsequence. A method of assaying for a target nucleic acid in a sample,comprises: contacting the sample to a complex comprising a guide nucleicacid comprising a segment that is reverse complementary to a segment ofthe target nucleic acid and a programmable nuclease that exhibitssequence independent cleavage upon forming a complex comprising thesegment of the guide nucleic acid binding to the segment of the targetnucleic acid, wherein the sample comprises at least one nucleic acidcomprising at least 50% sequence identity to the segment of the targetnucleic acid; and assaying for cleavage of at least one detector nucleicacids of a population of detector nucleic acids, wherein the cleavageindicates a presence of the target nucleic acid in the sample andwherein absence of the cleavage indicates an absence of the targetnucleic acid in the sample.

The methods of assaying for a target nucleic acid described herein mayfurther comprise introducing a PAM sequence into a target nucleic acidsegment that lacks a PAM sequence. For example, a method of assaying fora target nucleic acid segment in a sample, wherein the target nucleicacid segment lacks a PAM sequence, comprises amplifying the targetnucleic acid segment using a primer having a region that is reversecomplementary to the target nucleic acid segment and a region that has aPAM sequence reverse complement, thereby generating a PAM target nucleicacid having a PAM sequence adjacent to target sequence of anamplification product; contacting the PAM target nucleic acid toPAM-dependent sequence specific nuclease complex comprising a guidenucleic acid and a programmable nuclease that exhibits sequenceindependent cleavage upon forming a complex comprising the segment ofthe guide nucleic acid binding to the segment of the PAM target nucleicacid; and assaying for cleavage of at least one detector nucleic acid ofa population of detector nucleic acids, wherein the cleavage indicates apresence of the target nucleic acid in the sample and wherein theabsence of the cleavage indicates an absence of the target nucleic acidin the sample. A PAM-dependent sequence specific nuclease, often, is aprogrammable nuclease. Sometimes, a PAM-dependent sequence specificnuclease is a PAM-dependent sequence specific endonuclease.

A number of reagents are consistent with the compositions and methodsdisclosed herein. The reagents described herein for detecting a disease,cancer, or genetic disorder comprise a guide nucleic acid targeting thetarget nucleic acid segment indicative of a disease, cancer, or geneticdisorder. The reagents disclosed herein can include programmablenucleases, guide nucleic acids, target nucleic acids, and buffers. Asdescribed herein, target nucleic acid comprising DNA or RNA may bedetected (e.g., the target DNA hybridizes to the guide nucleic) using aprogrammable nuclease and other reagents disclosed herein. As describedherein, target nucleic acids comprising DNA may be an amplicon of anucleic acid of interest and the amplicon can be detected (e.g., thetarget DNA hybridizes to the guide nucleic) using a programmablenuclease and other reagents disclosed herein. Additionally, detection ofmultiple target nucleic acids is possible using two or more programmablenucleases complexed to guide nucleic acids that target the multipletarget nucleic acids, wherein the programmable nucleases exhibitdifferent sequence-independent cleavage of the nucleic acid of areporter (e.g., cleavage of an RNA reporter by a first programmablenuclease and cleavage of a DNA reporter by a second programmablenuclease).

Programmable Nucleases

The programmable nucleases disclosed herein (e.g., a type V or VI CRISPRenzyme) enable the detection of target nucleic acids (e.g., DNA or RNA).Additionally, detection by a first programmable nuclease, which cancleave RNA reporters, allows for multiplexing with programmablenucleases, which cleave DNA reporters.

The detection of the target nucleic acid is facilitated by aprogrammable nuclease. A programmable nuclease can comprise aprogrammable nuclease capable of being activated when complexed with aguide nucleic acid and target nucleic acid. The programmable nucleasecan become activated after binding of a guide nucleic acid to a targetnucleic, in which the activated programmable nuclease can cleave thetarget nucleic acid and exhibits sequence-independent cleavage activity.Sequence-independent cleavage activity, also referred to herein as“trans cleavage activity” or “collateral cleavage activity”, can benon-specific cleavage of nearby single-stranded nucleic acids by theactivated programmable nuclease, such as trans cleavage of nucleic acidsin a reporter, where the reporter also comprises a detection moiety. Thereporter may comprise a detector nucleic acid. Once the nucleic acid ofthe reporter is cleaved by the activated programmable nuclease, thedetection moiety is released from the nucleic acid of the reporter, andgenerates a detectable signal. Often the detection moiety is at leastone of a fluorophore, a dye, a polypeptide, or a nucleic acid. Sometimesthe detection moiety binds to a capture molecule immobilized on a solidsurface. The detectable signal can be visualized on the solid surface toassess the presence, the absence, or level of presence of the targetnucleic acid. A detectable signal can be a calorimetric, potentiometric,amperometric, optical (e.g., fluorescent, colorometric, etc.), orpiezo-electric signal. Often, the detectable signal is present prior tocleavage of the nucleic acid of the reporter and changes upon cleavageof the nucleic acid of the reporter. Sometimes, the signal is absentprior to cleavage of the nucleic acid of the reporter and is presentupon cleavage of the nucleic acid of the reporter. The detectable signalcan be immobilized on a solid surface for detection. The programmablenuclease can be a DNA-activated programmable RNA nuclease, aDNA-activated programmable DNA nuclease, or an RNA-activatedprogrammable RNA nuclease. A DNA-activated programmable RNA nuclease isa programmable nuclease, which upon hybridization of its guide nucleicacid to a target DNA, exhibits sequence-independent cleavage of areporter having a RNA (an RNA reporter). A DNA-activated programmableDNA nuclease is a programmable nuclease, which upon hybridization of itsguide nucleic acid to a target DNA, exhibits sequence-independentcleavage of a reporter having a DNA (a DNA reporter). A RNA-activatedprogrammable RNA nuclease is a programmable nuclease, which uponhybridization of its guide nucleic acid to a target RNA, exhibitssequence-independent cleavage of a reporter having a RNA (a RNAreporter). The DNA-activated programmable DNA nuclease can be a Type VCRISPR/Cas enzyme (e.g., Cas12). The DNA-activated programmable RNAnuclease can be a Type VI CRISPR/Cas enzyme (e.g., Cas13). TheRNA-activated programmable RNA nuclease can be a Type VI CRISPR/Casenzyme (e.g., Cas13).

The programmable nucleases disclosed herein may elicit reporter activityupon cleavage of the nucleic acid of the reporter. Reporter activityrefers to transcollatoral cleavage activity of a detector nucleic acid.A reporter activity may be a calorimetric, potentiometric, amperometric,optical (e.g., fluorescent, colorometric, etc.), or piezo-electricsignal. For example, cleavage of the nucleic acid of the reporter by theprogrammable nuclease may elicity a fluorescent signal. A reporteractivity may increase or decrease over time in response to aprogrammable nuclease trans cleavage activity. A reporter activity mayaccumulate over time in response to a programmable nuclease transcleavage activity. A maximal reporter activity may occur when a reportersignal (e.g., a calorimetric, potentiometric, amperometric, optical(e.g., fluorescent, colorometric, etc.), or piezo-electric signal) ishighest within a designated assay. In some embodiments, a maximalreporter signal may occur when a reporter signal reaches a maximumsignal, after which the reporter signal decreases. In some embodiments,a maximal reporter signal may occur when a reporter signal increases tosaturation after which the signal is no longer increasing.

The programmable nucleases disclosed herein may exhibit cis-cleavageactivity or target cleavage activity. Target cleavage activity may referto the cleavage of a target nucleic acid by the programmable nuclease.

In some embodiments, the Type V CRISPR/Cas enzyme is a programmableCas12 nuclease. Type V CRISPR/Cas enzymes (e.g., Cas12 or Cas14) lack anHNH domain. A Cas12 nuclease of the present disclosure cleaves a nucleicacid via a single catalytic RuvC domain. This single catalytic RuvCdomain includes 3 partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III,also referred to herein as subdomains) that are not contiguous withrespect to the primary amino acid sequence of the Cas12 protein, butform an RuvC domain once the protein is produced and folds. In someembodiments, a programmable nuclease comprises three partial RuvCdomains. In some embodiments, a programmable nuclease comprises anRuvC-I subdomain, an RuvC-II subdomain, and an RuvC-III subdomain. TheRuvC domain is within a nuclease, or “NUC” lobe of the protein, and theCas12 nucleases further comprise a recognition, or “REC” lobe. The RECand NUC lobes are connected by a bridge helix and the Cas12 proteinsadditionally include two domains for PAM recognition termed the PAMinteracting (PI) domain and the wedge (WED) domain. (Murugan et al., MolCell. 2017 Oct. 5; 68(1): 15-25). In some embodiments, a Cas12 protein(e.g., a programmable nuclease having a sequence with at least 60%sequence identity to SEQ ID NO: 11) may recognize a PAM having asequence of YYN, where N represents any nucleic acid and (e.g., A, T, C,G, or U) Y represents any pyrimidine (e.g., C or T). In someembodiments, a Cas12 protein may recongnize a PAM having a sequence ofYR, where Y represents any pyrimidine (e.g., C or T) and R representsany purine (e.g., A or G). A programmable Cas12 nuclease can be a Cas12a(also referred to as Cpf1) protein, a Cas12b protein, Cas12c protein,Cas12d protein (also referred to as a CasY protein), or a Cas12eprotein. For example, the programmable Cas12 nuclease may be a Cas12a. Aprogrammable Cas12 nuclease can be a Cas12 variant. In some cases, asuitable Cas12 protein comprises an amino acid sequence having at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 92%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto any one of the Cas12 proteins or Cas12 variants provided in TABLE 1(e.g., any one of SEQ ID NO: 1-SEQ ID NO: 11, SEQ ID NO: 282, or SEQ IDNO: 571-SEQ ID NO: 602). For example, a suitable Cas12 protein comprisesa sequence with at least 60% sequence identity to SEQ ID NO: 11. In someembodiments, a suitable Cas12 protein comprises a sequence with at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%,amino acid sequence identity to SEQ ID NO: 11. In some embodiments, asuitable Cas12 protein comprises a sequence with at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 92%, at least95%, at least 97%, at least 98%, at least 99%, or 100%, amino acidsequence identity to SEQ ID NO: 1. For example, a suitable Cas12 proteinmay comprise an amino acid sequence having at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 92%, at least 95%, at least 97%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to SEQ IDNO: 11. A Cas12 protein can have a sequence as set forth in SEQ ID NO:11. In some embodiments, a Cas12 nuclease may have at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 95%, at least 97%, at least 99%, or 100% sequence identity to anyone of SEQ ID NO: 1-SEQ ID NO: 11, SEQ ID NO: 282, or SEQ ID NO: 571-SEQID NO: 602.

TABLE 1 Cas12 Sequences SEQ ID NO Description Sequence SEQ Lachno-MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGV ID spiraceaeKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLR NO: bacteriumKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGF 1 ND2006FDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIK (LbCas12a)EKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKI AISNKEWLEYAQTSVKH SEQAcidamino- MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELK IDcoccus sp. PIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATY NO: BV316RNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTE 2 (AsCas12a)HENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN SEQ FrancisellaMSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAK ID novicidaQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKD NO: U112TIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKA 3 (FnCas12a)NSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN SEQ Porphyro-MKTQHFFEDFTSLYSLSKTIRFELKPIGKTLENIKKNGLIRRDEQRLDDYEK ID monasLKKVIDEYHEDFIANILSSFSFSEEILQSYIQNLSESEARAKIEKTMRDTLAK NO: macacaeAFSEDERYKSIFKKELVKKDIPVWCPAYKSLCKKFDNFTTSLVPFHENRK 4 (PmCas12a)NLYTSNEITASIPYRIVHVNLPKFIQNIEALCELQKKMGADLYLEMMENLRNVWPSFVKTPDDLCNLKTYNHLMVQSSISEYNRFVGGYSTEDGTKHQGINEWINIYRQRNKEMRLPGLVFLHKQILAKVDSSSFISDTLENDDQVFCVLRQFRKLFWNTVSSKEDDAASLKDLFCGLSGYDPEAIYVSDAHLATISKNIFDRWNYISDAIRRKTEVLMPRKKESVERYAEKISKQIKKRQSYSLAELDDLLAHYSEESLPAGFSLLSYFTSLGGQKYLVSDGEVILYEEGSNIWDEVLIAFRDLQVILDKDFTEKKLGKDEEAVSVIKKALDSALRLRKFFDLLSGTGAEIRRDSSFYALYTDRMDKLKGLLKMYDKVRNYLTKKPYSIEKFKLHFDNPSLLSGWDKNKELNNLSVIFRQNGYYYLGIMTPKGKNLFKTLPKLGAEEMFYEKMEYKQIAEPMLMLPKVFFPKKTKPAFAPDQSVVDIYNKKTFKTGQKGFNKKDLYRLIDFYKEALTVHEWKLFNFSFSPTEQYRNIGEFFDEVREQAYKVSMVNVPASYIDEAVENGKLYLFQIYNKDFSPYSKGIPNLHTLYWKALFSEQNQSRVYKLCGGGELFYRKASLHMQDTTVHPKGISIHKKNLNKKGETSLFNYDLVKDKRFTEDKFFFHVPISINYKNKKITNVNQMVRDYIAQNDDLQIIGIDRGERNLLYISRIDTRGNLLEQFSLNVIESDKGDLRTDYQKILGDREQERLRRRQEWKSIESIKDLKDGYMSQVVHKICNMVVEHKAIVVLENLNLSFMKGRKKVEKSVYEKFERMLVDKLNYLVVDKKNLSNEPGGLYAAYQLTNPLFSFEELHRYPQSGILFFVDPWNTSLTDPSTGFVNLLGRINYTNVGDARKFFDRFNAIRYDGKGNILFDLDLSRFDVRVETQRKLWTLTTFGSRIAKSKKSGKWMVERIENLSLCFLELFEQFNIGYRVEKDLKKAILSQDRKEFYVRLIYLFNLMMQIRNSDGEEDYILSPALNEKNLQFDSRLIEAKDLPVDADANGAYNVARKGLMVVQRIKRGDHESIHRIGRAQWLRYVQEGIVE SEQ MoraxellaMLFQDFTHLYPLSKTVRFELKPIDRTLEHIHAKNFLSQDETMADMHQKVK ID bovoculiVILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDDELQKQLKDL NO: 237QAVLRKEIVKPIGNGGKYKAGYDRLFGAKLFKDGKELGDLAKFVIAQEG 5 (MbCas12a)ESSPKLAHLAHFEKFSTYFTGFHDNRKNMYSDEDKHTAIAYRLIHENLPRFIDNLQILTTIKQKHSALYDQIINELTASGLDVSLASHLDGYHKLLTQEGITAYNTLLGGISGEAGSPKIQGINELINSHEINQHCHKSERIAKLRPLHKQILSDGMSVSFLPSKFADDSEMCQAVNEFYRHYADVFAKVQSLFDGFDDHQKDGIYVEHKNLNELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSLASLEQAIEHYTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSGKNPEMTQLRQLKELLDNALNVAHFAKLLTTKTTLDNQDGNFYGEFGVLYDELAKIPTLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLALLDKAHKKVFDNAPNTGKSIYQKMIYKYLEVRKQFPKVFFSKEAIAINYHPSKELVEIKDKGRQRSDDERLKLYRFILECLKIHPKYDKKFEGAIGDIQLFKKDKKGREVPISEKDLFDKINGIFSSKPKLEMEDFFIGEFKRYNPSQDLVDQYNIYKKIDSNDNRKKENFYNNHPKFKKDLVRYYYESMCKHEEWEESFEFSKKLQDIGCYVDVNELFTEIETRRLNYKISFCNINADYIDELVEQGQLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLADPIYKLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDEVNVIGIDRGERHLLYLTVINSKGEILEQCSLNDITTASANGTQMTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQISQLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNADKDYFEFHIDYAKFTDKAKNSRQIWTICSHGDKRYVYDKTANQNKGAAKGINVNDELKSLFARHHINEKQPNLVMDICQNNDKEFHKSLMYLLKTLLALRYSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGLWLLNELKNSDDL NKVKLAIDNQTWLNFAQNRSEQ Moraxella MGIHGVPAALFQDFTHLYPLSKTVRFELKPIGRTLEHIHAKNFLSQDETMA IDbovoculi DMYQKVKVILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDDG NO: AAX08_00LQKQLKDLQAVLRKESVKPIGSGGKYKTGYDRLFGAKLFKDGKELGDLA 6 205KFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMYSDEDKHTAIAYR (Mb2Cas12LIHENLPRFIDNLQILTTIKQKHSALYDQIINELTASGLDVSLASHLDGYHK a)LLTQEGITAYNRIIGEVNGYTNKHNQICHKSERIAKLRPLHKQILSDGMGVSFLPSKFADDSEMCQAVNEFYRHYTDVFAKVQSLFDGFDDHQKDGIYVEHKNLNELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSLASLEQAIEHEITARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSGKNPEMTQLRQLKELLDNALNVAHFAKLLTTKTTLDNQDGNFYGEFGVLYDELAKIPTLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLALLDKAHKKVFDNAPNTGKNVYQKMVYKLLPGPNKMLPKVFFAKSNLDYYNPSAELLDKYAKGTHKKGDNFNLKDCHALIDFFKAGINKHPEWQHFGFKFSPTSSYRDLSDFYREVEPQGYQVKFVDINADYIDELVEQGKLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLADPIYKLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDEVNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGTQVTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQINQLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNTDKGYFEFHIDYAKFTDKAKNSRQKWAICSHGDKRYVYDKTANQNKGAAKGINVNDELKSLFARYHINDKQPNLVMDICQNNDKEFHKSLMCLLKTLLALRYSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGLWLLNELKNSDDLNKV KLAIDNQTWLNFAQNR SEQMoraxella MGIHGVPAALFQDFTHLYPLSKTVRFELKPIGKTLEHIHAKNFLNQDETM ID bovoculiADMYQKVKAILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDD NO: AAX11_00GLQKQLKDLQAVLRKEIVKPIGNGGKYKAGYDRLFGAKLFKDGKELGDL 7 205AKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMYSDEDKHTAIAY (Mb3Cas12RLIHENLPRFIDNLQILATIKQKHSALYDQIINELTASGLDVSLASHLDGYH a)KLLTQEGITAYNTLLGGISGEAGSRKIQGINELINSFIHNQHCHKSERIAKLRPLHKQILSDGMGVSFLPSKFADDSEVCQAVNEFYRHYADVFAKVQSLFDGFDDYQKDGIYVEYKNLNELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSLASLEQAIEHYTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSDKSPEIRQLKELLDNALNVAHFAKLLTTKTTLHNQDGNFYGEFGALYDELAKIATLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLALLDKAHKKVFDNAPNTGKSVYQKMIYKLLPGPNKMLPKVFFAKSNLDYYNPSAELLDKYAQGTHKKGDNFNLKDCHALIDFFKAGINKHPEWQHFGFKFSPTSSYQDLSDFYREVEPQGYQVKFVDINADYINELVEQGQLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLVNPIYKLNGEAEIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDEVNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGTQMTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQISQLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNADRGYFEFHIDYAKFNDKAKNSRQIWKICSHGDKRYVYDKTANQNKGATIGVNVNDELKSLFTRYHINDKQPNLVMDICQNNDKEFHKSLMYLLKTLLALRYSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGLWLLNELKNSDDLNKVKLAIDNQTWLNFAQNR SEQ Thiomicro-MGIHGVPAATKTFDSEFFNLYSLQKTVRFELKPVGETASFVEDFKNEGLK ID spira sp. XS5RVVSEDERRAVDYQKVKEIIDDYHRDFIEESLNYFPEQVSKDALEQAFHL NO: (TsCas12a)YQKLKAAKVEEREKALKEWEALQKKLREKVVKCFSDSNKARFSRIDKKE 8LIKEDLINWLVAQNREDDIPTVETFNNFTTYFTGFHENRKNIYSKDDHATAISFRLIHENLPKFFDNVISFNKLKEGFPELKFDKVKEDLEVDYDLKHAFEIEYFVNFVTQAGIDQYNYLLGGKTLEDGTKKQGMNEQINLFKQQQTRDKARQIPKLIPLFKQILSERTESQSFIPKQFESDQELFDSLQKLHNNCQDKFTVLQQAILGLAEADLKKVFIKTSDLNALSNTIFGNYSVFSDALNLYKESLKTKKAQEAFEKLPAHSIHDLIQYLEQFNSSLDAEKQQSTDTVLNYFIKTDELYSRFIKSTSEAFTQVQPLFELEALSSKRRPPESEDEGAKGQEGFEQIKRIKAYLDTLMEAVHFAKPLYLVKGRKMIEGLDKDQSFYEAFEMAYQELESLIIPIYNKARSYLSRKPFKADKFKINFDNNTLLSGWDANKETANASILFKKDGLYYLGIMPKGKTFLFDYFVSSEDSEKLKQRRQKTAEEALAQDGESYFEKIRYKLLPGASKMLPKVFFSNKNIGFYNPSDDILRIRNTASHTKNGTPQKGHSKVEFNLNDCHKMIDFFKSSIQKHPEWGSFGFTFSDTSDFEDMSAFYREVENQGYVISFDKIKETYIQSQVEQGNLYLFQIYNKDFSPYSKGKPNLHTLYWKALFEEANLNNVVAKLNGEAEIFFRRHSIKASDKVVHPANQAIDNKNPHTEKTQSTFEYDLVKDKRYTQDKFFFHVPISLNFKAQGVSKFNDKVNGFLKGNPDVNIIGIDRGERHLLYFTVVNQKGEILVQESLNTLMSDKGHVNDYQQKLDKKEQERDAARKSWTTVENIKELKEGYLSHVVHKLAHLIIKYNAIVCLEDLNFGFKRGRFKVEKQVYQKFEKALIDKLNYLVFKEKELGEVGHYLTAYQLTAPFESFKKLGKQSGILFYVPADYTSKIDPTTGFVNFLDLRYQSVEKAKQLLSDFNAIRFNSVQNYFEFEIDYKKLTPKRKVGTQSKWVICTYGDVRYQNRRNQKGHWETEEVNVTEKLKALFASDSKTTTVIDYANDDNLIDVILEQDKASFFKELLWLLKLTMTLRHSKIKSEDDFILSPVKNEQGEFYDSRKAGEVWPKDADANGAYHIALKGLWNLQQINQWEKGKTLNLAIKNQDWFSFIQEKPYQE SEQ ButyrivibrioMGIHGVPAAYYQNLTKKYPVSKTIRNELIPIGKTLENIRKNNILESDVKRK ID sp. NC3005QDYEHVKGIMDEYHKQLINEALDNYMLPSLNQAAEIYLKKHVDVEDREE NO: (BsCas12a)FKKTQDLLRREVTGRLKEHENYTKIGKKDILDLLEKLPSISEEDYNALESF 9RNFYTYFTSYNKVRENLYSDEEKSSTVAYRLINENLPKFLDNIKSYAFVKAAGVLADCIEEEEQDALFMVETFNMTLTQEGIDMYNYQIGKVNSAINLYNQKNHKVEEFKKIPKMKVLYKQILSDREEVFIGEFKDDETLLSSIGAYGNVLMTYLKSEKINIFFDALRESEGKNVYVKNDLSKTTMSNIVFGSWSAFDELLNQEYDLANENKKKDDKYFEKRQKELKKNKSYTLEQMSNLSKEDISPIENYIERISEDIEKICIYNGEFEKIVVNEHDSSRKLSKNIKAVKVIKDYLDSIKELEHDIKLINGSGQELEKNLVVYVGQEEALEQLRPVDSLYNLTRNYLTKKPFSTEKVKLNFNKSTLLNGWDKNKETDNLGILFFKDGKYYLGIMNTTANKAFVNPPAAKTENVFKKVDYKLLPGSNKMLPKVFFAKSNIGYYNPSTELYSNYKKGTHKKGPSFSIDDCHNLIDFFKESIKKHEDWSKFGFEFSDTADYRDISEFYREVEKQGYKLTFTDIDESYINDLIEKNELYLFQIYNKDFSEYSKGKLNLHTLYFMMLFDQRNLDNVVYKLNGEAEVFYRPASIAENELVIHKAGEGIKNKNPNRAKVKETSTFSYDIVKDKRYSKYKFTLHIPITMNFGVDEVRRFNDVINNALRTDDNVNVIGIDRGERNLLYVVVINSEGKILEQISLNSIINKEYDIETNYHALLDEREDDRNKARKDWNTIENIKELKTGYLSQVVNVVAKLVLKYNAIICLEDLNFGFKRGRQKVEKQVYQKFEKMLIEKLNYLVIDKSREQVSPEKMGGALNALQLTSKFKSFAELGKQSGIIYYVPAYLTSKIDPTTGFVNLFYIKYENIEKAKQFFDGFDFIRFNKKDDMFEFSFDYKSFTQKACGIRSKWIVYTNGERIIKYPNPEKNNLFDEKVINVTDEIKGLFKQYRIPYENGEDIKEIIISKAEADFYKRLFRLLHQTLQMRNSTSDGTRDYIISPVKNDRGEFFCSEFSEGTMPKDADANGAYNIARKGLWVLEQIRQKDEGEKVNLSMTNAEWLKYAQLH LL SEQ AacCas12bMAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENL IDYRRSPNGDGEQECDKTAEECKAELLERLRARQVENGHRGPAGSDDELLQ NO:LARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKP 10RWVRMREAGEPGWEEEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKLVEQKNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELINQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADDLIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKVFAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMVNQRIEGYLVKQIRSRVPLQDSACENTGDI SEQ Cas12MKKIDNFVGCYPVSKTLRFKAIPIGKTQENIEKKRLVEEDEVRAKDYKAV ID VariantKKLIDRYHREFIEGVLDNVKLDGLEEYYMLFNKSDREESDNKKIEIMEERF NO:RRVISKSFKNNEEYKKIFSKKIIEEILPNYIKDEEEKELVKGFKGFYTAFVG 11YAQNRENMYSDEKKSTAISYRIVNENMPRFITNIKVFEKAKSILDVDKINEINEYILNNDYYVDDFFNIDFFNYVLNQKGIDIYNAIIGGIVTGDGRKIQGLNECINLYNQENKKIRLPQFKPLYKQILSESESMSFYIDEIESDDMLIDMLKESLQIDSTINNAIDDLKVLFNNIFDYDLSGIFINNGLPITTISNDVYGQWSTISDGWNERYDVLSNAKDKESEKYFEKRRKEYKKVKSFSISDLQELGGKDLSICKKINEIISEMIDDYKSKIEEIQYLFDIKELEKPLVTDLNKIELIKNSLDGLKRIERYVIPFLGTGKEQNRDEVFYGYFIKCIDAIKEIDGVYNKTRNYLTKKPYSKDKFKLYFENPQLMGGWDRNKESDYRSTLLRKNGKYYVAIIDKSSSNCMMNIEEDENDNYEKINYKLLPGPNKMLPKVFFSKKNREYFAPSKEIERIYSTGTFKKDTNFVKKDCENLITFYKDSLDRHEDWSKSFDFSFKESSAYRDISEFYRDVEKQGYRVSFDLLSSNAVNTLVEEGKLYLFQLYNKDFSEKSHGIPNLHTMYFRSLFDDNNKGNIRLNGGAEMFMRRASLNKQDVTVHKANQPIKNKNLLNPKKTTTLPYDVYKDKRFTEDQYEVHIPITMNKVPNNPYKINHMVREQLVKDDNPYVIGIDRGERNLIYVVVVDGQGHIVEQLSLNEIINENNGISIRTDYHTLLDAKERERDESRKQWKQIENIKELKEGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLITKLNYMVDKKKDYNKPGGVLNGYQLTTQFESFSKMGTQNGIMFYIPAWLTSKMDPTTGFVDLLKPKYKNKADAQKFFSQFDSIRYDNQEDAFVFKVNYTKFPRTDADYNKEWEIYTNGERIRVFRNPKKNNEYDYETVNVSERMKELFDSYDLLYDKGELKETICEMEESKFFEELIKLFRLTLQMRNSISGRTDVDYLISPVKNSNGYFYNSNDYKKEGAKYPKDADANGAYNIARKVLWAIEQFKMADEDKLDKTKISIKNQE WLEYAQTHCE SEQ CasY3MKAKKSFYNQKRKFGKRGYRLHDERIAYSGGIGSMRSIKYELKDSYGIAG IDLRNRIADATISDNKWLYGNINLNDYLEWRSSKTDKQIEDGDRESSLLGFW NO:LEALRLGFVFSKQSHAPNDFNETALQDLFETLDDDLKHVLDRKKWCDFIK 282IGTPKTNDQGRLKKQIKNLLKGNKREEIEKTLNESDDELKEKINRIADVFAKNKSDKYTIFKLDKPNTEKYPRINDVQVAFFCHPDFEEITERDRTKTLDLIINRFNKRYEITENKKDDKTSNRMALYSLNQGYIPRVLNDLFLFVKDNEDDFSQFLSDLENFFSFSNEQIKIIKERLKKLKKYAEPIPGKPQLADKWDDYASDFGGKLESWYSNRIEKLKKIPESVSDLRNNLEKIRNVLKKQNNASKILELSQKIIEYIRDYGVSFEKPEIIKFSWINKTKDGQKKVFYVAKMADREFIEKLDLWMADLRSQLNEYNQDNKVSFKKKGKKIEELGVLDFALNKAKKNKSTKNENGWQQKLSESIQSAPLFFGEGNRVRNEEVYNLKDLLFSEIKNVENILMSSEAEDLKNIKIEYKEDGAKKGNYVLNVLARFYARFNEDGYGGWNKVKTVLENIAREAGTDFSKYGNNNNRNAGRFYLNGRERQVFTLIKFEKSITVEKILELVKLPSLLDEAYRDLVNENKNHKLRDVIQLSKTIMALVLSHSDKEKQIGGNYIHSKLSGYNALISKRDFISRYSVQTTNGTQCKLAIGKGKSKKGNEIDRYFYAFQFFKNDDSKINLKVIKNNSHKNIDFNDNENKINALQVYSSNYQIQFLDWFFEKHQGKKTSLEVGGSFTIAEKSLTIDWSGSNPRVGFKRSDTEEKRVFVSQPFTLIPDDEDKERRKERMIKTKNRFIGIDIGEYGLAWSLIEVDNGDKNNRGIRQLESGFITDNQQQVLKKNVKSWRQNQIRQTFTSPDTKIARLRESLIGSYKNQLESLMVAKKANLSFEYEVSGFEVGGKRVAKIYDSIKRGSVRKKDNNSQNDQSWGKKGINEWSFETTAAGTSQFCTHCKRWSSLAIVDIEEYELKDYNDNLFKVKINDGEVRLLGKKGWRSGEKIKGKELFGPVKDAMRPNVDGLGMKIVKRKYLKLDLRDWVSRYGNMAIFICPYVDCHHISHADKQAAFNIAVRGYLKSVNPDRAIKHGDKGLSRDFLCQEEGKLNFEQIGLLI SEQ Cas12MATLVSFTKQYQVQKTLRFELIPQGKTQANIDAKGFINDDLKRDENYMK ID variantVKGVIDELHKNFIEQTLVNVDYDWRSLATAIKNYRKDRSDTNKKNLEKT NO:QEAARKEIIAWFEGKRGNSAFKNNQKSFYGKLFKKELFSEILRSDDLEYDE 571ETQDAIACFDKFTTYFVGFHENRKNMYSTEAKSTSVAYRVVNENFSKFLSNCEAFSVLEAVCPNVLVEAEQELHLHKAFSDLKLSDVFKVEAYNKYLSQTGIDYYNQIIGGISSAEGVRKIRGVNEVVNNAIQQNDELKVALRNKQFTMVQLFKQILSDRSTLSFVSEQFTSDQEVITVVKQFNDDIVNNKVLAVVKTLFENFNSYDLEKIYINSKELASVSNALLKDWSKIRNAVLENKIIELGANPPKTKISAVEKEVKNKDFSIAELASYNDKYLDKEGNDKEICSIANVVLEAVGALEIMLAESLPADLKTLENKNKVKGILDAYENLLHLLNYFKVSAVNDVDLAFYGAFEKVYVDISGVMPLYNKVRNYATKKPYSVEKFKLNFAMPTLADGWDKNKERDNGSIILLKDGQYYLGVMNPQNKPVIDNAVCNDAKGYQKMVYKMFPEISKMVTKCSTQLNAVKAHFEDNTNDFVLDDTDKFISDLTITKEIYDLNNVLYDGKKKFQIDYLRNTGDFAGYHKALETWIDFVKEFLSKYRSTAIYDLTTLLPTNYYEKLDVFYSDVNNLCYKIDYENISVEQVNEWVEEGNLYLFKIYNKDFATGSTGKPNLHTMYWNAVFAEENLHDVVVKLNGGAELFYRPKSNMPKVEHRVGEKLVNRKNVNGEPIADSVHKEIYAYANGKISKSELSENAQEELPLAIIKDVKHNITKDKRYLSDKYFFHVPITLNYKANGNPSAFNTKVQAFLKNNPDVNIIGIDRGERNLLYVVVIDQQGNIIDKKQVSYNKVNGYDYYEKLNQREKERIEARQSWGAVGKIKELKEGYLSLVVREIADMMVKYNAIVVMENLNAGFKRVRGGIAEKAVYQKFEKMLIDKLNYLVFKDVEAKEAGGVLNAYQLTDKFDSFEKMGNQSGFLFYVPAAYTSKIDPVTGFANVFSTKHITNTEAKKEFICSFNSLRYDEAKDKFVLECDLNKFKIVANSHIKNWKFIIGGKRIVYNSKNKTYMEKYPCEDLKATLNASGIDFSSSEIINLLKNVPANREYGKLFDETYWAIMNTLQMRNSNALTGEDYIISAVADDNEKVFDSRTCGAELPKDADANGAYHIALKGLYLLQRIDISEEGEKVDLSIKNEEWFKFVQQKEYA R SEQ Cas12MKEQFINRYPLSKTLRFSLIPVGETENNFNKNLLLKKDKQRAENYEKVKC ID variantYIDRFHKEYIESVLSKARIEKVNEYANLYWKSNKDDSDIKAMESLENDMR NO:KQISKQLTSTEIYKKRLFGKELICEDLPSFLTDKDERETVECFRSFTTYFKG 572FNTNRENMYSSDGKSTAIAYRCINDNLPRFLDNVKSFQKVFDNLSDETITKLNTDLYNIFGRNIEDIFSVDYFEFVLTQSGIEIYNSMIGGYTCSDKTKIQGLNECINLYNQQVAKNEKSKKLPLMKPLYKQILSEKDSVSFIPEKFNSDNEVLHAIDDYYTGHIGDFDLLTELLQSLNTYNANGIFVKSGVAITDISNGAFNSWNVLRSAWNEKYEALHPVTSKTKIDKYIEKQDKIYKAIKSFSLFELQSLGNENGNEITDWYISSINESNSKIKEAYLQAQKLLNSDYEKSYNKRLYKNEKATELVKNLLDAIKEFQKLIKPLNGTGKEENKDELFYGKFTSYYDSIADIDRLYDKVRNYITQKPYSKDKIKLNFDNPQLLGGWDKNKESDYRTVLLHKDGLYYLAVMDKSHSKAFVDAPEITSDDKDYYEKMEYKLLPGPNKMLPKVFFASKNIDTFQPSDRILDIRKRESFKKGATFNKAECHEFIDYFKDSIKKHDDWSQFGFKFSPTESYNDISEFYREISDQGYSVRFNKISKNYIDGLVNNGYIYLFQIYNKDFSKYSKGTPNLHTLYFKMLFDERNLSNVVYKLNGEAEMFYREASIGDKEKITHYANQPIKNKNPDNEKKESVFEYDIVKDKRFTKRQFSLHLPITINFKAHGQEFLNYDVRKAVKYKDDNYVIGIDRGERNLIYISVINSNGEIVEQMSLNEIISDNGHKVDYQKLLDTKEKERDKARKNWTSVENIKELKEGYISQVVHKICELVIKYDAVIAMEDLNFGFKRGRFPVEKQVYQKFENMLISKLNLLIDKKAEPTEDGGLLRAYQLTNKFDGVNKAKQNGIIFYVPAWDTSKIDPATGFVNLLKPKCNTSVPEAKKLFETIDDIKYNANTDMFEFYIDYSKFPRCNSDFKKSWTVCTNSSRILTFRNKEKNNKWDNKQIVLTDEFKSLFNEFGIDYKGNLKDSILSISNADFYRRLIKLLSLTLQMRNSITGSTLPEDDYLISPVANKSGEFYDSRNYKGTNAALPCDADANGAYNIARKALWAINVLKDTPDDMLNKA KLSITNAEWLEYTQK SEQCas12 MNNPRGAFGGFTNLYSLSKTLRFELKPYLEIPEGEKGKLFGDDKEYYKNC ID variantKTYTEYYLKKANKEYYDNEKVKNTDLQLVNFLHDERIEDAYQVLKPVFD NO:TLHEEFITDSLESAEAKKIDFGNYYGLYEKQKSEQNKDEKKKIDKPLETER 573GKLRKAFTPIYEAEGKNLKNKAGKEKKDKDILKESGFKVLIEAGILKYIKNNIDEFADKKLKNNEGKEITKKDIETALGAENIEGIFDGFFTYFSGFNQNRENYYSTEEKATAVASRIVDENLSKFCDNILLYRKNENDYLKIFNFLKNKGKDLKLKNSKFGKENEPEFIPAYDMKNDEKSFSVADFVNCLSQGEIEKYNAKIANANYLINLYNQNKDGNSSKLSMFKILYKQIGCGEKKDFIKTIKDNAELKQILEKACEAGKKYFIRGKSEDGGVSNIFDFTDYIQSHENYKGVYWSDKAINTISGKYFANWDTLKNKLGDAKVFNKNTGEDKADVKYKVPQAVMLSELFAVLDDNAGEDWREKGIFFKASLFEGDQNKSEIIKNANRPSQALLKMICDDMESLAKNFIDSGDKILKISDRDYQKDENKQKIKNWLDNALWINQILKYFKVKANKIKGDSIDARIDSGLDMLVFSSDNPAEDYDMIRNYLTQKPQDEINKLKLNFENSSLAGGWDENKEKDNSCIILKDEQDKQYLAVMKYENTKVFEQKNSQLYIADNAAWKKMIYKLVPGASKTLPKVFFSKKWTANRPTPSDIVEIYQKGSFKKENVDFNDKKEKDESRKEKNREKIIAELQKTCWMDIRYNIDGKIESAKYVNKEKLAKLIDFYKENLKKYPSEEESWDRLFAFGFSDTKSYKSIDQFYIEVDKQGYKLEFVTINKARLDEYVRDGKIYLFEIRSRDNNLVNGEEKTSAKNLQTIYWNAAFGGDDNKPKLNGEAEIFYRPAIAENKLNKKKDKNGKEIIDGYRFSKEKFIFHCPITLNFCLKETKINDKLNAALAKPENGQGVYFLGIDRGEKHLAYYSLVNQKGEILEQGTLNLPFLDKNGKSRSIKVEKKSFEKDSNGGIIKDKDGNDKIKIEFVECWNYNDLLDARAGDRDYARKNWTTIGTIKELKDGYISQVVRKIVDLSIYKNTETKEFREMPAFIVLEDLNIGFKRGRQKIEKQVYQKLELALAKKLNFLVDKKADIGEIGSVTKAIQLTPPVNNFGDMENRKQFGNMLYIRADYTSQTDPATGWRKSIYLKSGSESNVKEQIEKSFFDIRYESGDYCFEYRDRHGKMWQLYSSKNGVSLDRFHGERNNSKNVWESEKQPLNEMLDILFDEKRFDKSKSLYEQMFKGGVALTRLPKEINKKDKPAWESLRFVIILIQQIRNTGKNGDDRNGDFIQSPVRDEKTGEHFDSRIYLDKEQKGEKADLPTSGDANGAYNIARKGIVVAEHIKRGFDKLYISDEEWDTWLAGDEIW DKWLKENRESLTKTRK SEQCas12 MNGNRIIVYREFVGVTPVAKTLRNELRPIGHTQEHIIHNGLIQEDELRQEKS ID variantTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSSPSKDNKKALE NO:KEQSKMREQICTHMQSDSNYKNIFNAKFLKEILPDFIKNYNQYDAKDKAG 574KLETLALFNGFSTYFTDFFEKRKNVFTKEAVSTSIAYRIVHENSLTFLANMTSYKKISEKALDEIEVIEKNNQDKMGDWELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTKNNYNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETEKGNIIGKLKDIVNKYDELDEKRIYISKDFYETLSCFMSGNWNLITGCVENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDEKERNEFKNSNAKQYIREISNIITDTETAHLEYDEHISLIESEEKADEMKKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVPLYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILIRDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGANKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLIDYFKNSIEKHAEWRKYEFKFSATDSYNDISEFYREVEMQGYRIDWTYISEADINKLDEEGKIYLFQIYNKDFAENSTGKENLHTMYFKNIFSEENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQLDNGDVVRIPIPDDIYNEIYKMYNGYIKENDLSEAAKEYLDKVEVRTAQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMAVKYIAQNDDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVEKEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLMVEYNAIIAMEDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKGKSVDEPGGLLKGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAFNFKSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMSKTQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLEDNEINYADGHDVRIDMEKMDEDKNSEFFAQLLSLYKLTVQMRNSYTEAEEQEKGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYCIALKGLYEVLKIKSEWTEDGF DRNCLKLPHAEWLDFIQNKRYESEQ Cas12 MKKIDSFVNYYPLSKTLRFSLIPVGKTEDNFNAKLLLEEDEKRAIEYEKVK ID variantRYIDRYHKHFIETVLANFHLDDVNEYAELYYKAGKDDKDLKYMEKLEG NO:KMRKSISAAFTKDKKYKEIFGQEIIKNILPEFLENEDEKESVKMFQGFFTYF 575TGFNDNRKNMYTHEAQTTAISYRCINENLPKFLDNVQSFAKIKESISSDIMNKLDEVCMDLYGVYAQDMFCTDYFSFVLSQSGIDRYNNIIGGYVDDKGVKIQGINEYINLYNQQVDEKNKRLPLMKKLYKQILIEKESISFIPEKFESDNIVINAISDYYHNNVENLFDDFNKLFNEFSEYDDNGIFVTSGLAVTDISNAVFGSWNIISDSWNEEYKDSHPMKKTTNAEKYYEDMKKEYKKNLSFTIAELQRLGEAGCNDECKGDIKEYYKTTVAEKIENIKNAYEISKDLLASDYEKSNDKKLCKNDSAISLLKNLLDSIKDLEKTIKPLLGTGKEENKDDVFYGKFTNLYEMISEIDRLYDKVRNYVTQKPYSKDKIKLNFENPQHLGGWDKNKERDYRSVLLKKEDKYYLAIMDKSNNKAFIDFPDDGECYEKIEYKLLPGPNKMLPKVFFASSNIEYFAPSKKILEIRSRESFKKGDMFNLKDCHEFIDFFKESIKKHEDWSQFGFEFSPTEKYNDISEFYNEVKIQGYSLKYKNVSKKYIDELIECGQLYLFQIYNKDFSVYAKGNPNLHTMYFKMLFDERNLANVVYQLNGGAEMFYRKASIKDSEKIVHHANQPIKNKNADNVKKESVFEYDIIKDKRFTKRQFSIHIPITLNFKAKGQNFINNDVRMALKKADENYVIGIDRGERNLLYICVINSKGEIVEQKSLNEIIGDNGYRVDYHKLLDKKEAERDEARKSWGTIENIKELKEGYLSQIVHEISKLVIKYDAVIAIEDLNSGFKKGRFKVEKQVYQKFENMLCTKLNYLVDKNADANECGGLLKAYQLTNKEDGANRGRQNGIIFSVPAWLTSKIDPVTGFADLLRPKYKSVSESVEFISKIDNIRYNSKEDYFEFDIDYSKFPNSTASYKKKWTVCTYGERIINVRNKEKNNMWDNKTIVLTDEFKKLFADFGVDVSKNIKESVLAIDSKDFYYRFINLLANTLQLRNSEVGNVDVDYLISPVKGVDGSFYDSRLVKEKTLPENADANGAYNIARKALWAIDVLKQTKDEELKNA NLSIKNAEWLEYVQK SEQCas12 MRTMVTFEDFTKQYQVSKTLRFELIPQGKTLENMKRDGIISVDRQRNEDY ID variantQKAKGILDKLYKYILDFTMETVVIDWEALATATEEFRKSKDKKTYEKVQS NO:KIRTALLEHVKKQKVGTEDLFKGMFSSKIITGEVLAAFPEIRLSDEENLILE 576KFKDFTTYFTGFFENRKNVFTDEALSTSFTYRLVNDNFIKFFDNCIVFKNVVNISPHMAKSLETCASDLGIFPGVSLEEVFSISFYNRLLTQTGIDQFNQLLGGISGKEGEHKQQGLNEIINLAMQQNLEVKEVLKNKAHRFTPLFKQILSDRSTMSFIPDAFADDDEVLSAVDAYRKYLSEKNIGDRAFQLISDMEAYSPELMRIGGKYVSVLSQLLFYSWSEIRDGVKAYKESLITGKKTKKELENIDKEIKYGVTLQEIKEALPKKDIYEEVKKYAMSVVKDYHAGLAEPLPEKIETDDERASIKHIMDSMLGLYRFLEYFSHDSIEDTDPVFGECLDTILDDMNETVPLYNKVRNFSTRKVYSTEKFKLNFNNSSLANGWDKNKEQANGAILLRKEGEYFLGIFNSKNKPKLVSDGGAGIGYEKMIYKQFPDFKKMLPKCTISLKDTKAHFQKSDEDFTLQTDKFEKSIVITKQIYDLGTQTVNGKKKFQVDYPRLTGDMEGYRAALKEWIDFGKEFIQAYTSTAIYDTSLFRDSSDYPDLPSFYKDVDNICYKLTFEWIPDAVIDDCIDDGSLYLFKLHNKDFSSGSIGKPNLHTLYWKALFEEENLSDVVVKLNGQAELFYRPKSLTRPVVHEEGEVIINKTTSTGLPVPDDVYVELSKFVRNGKKGNLTDKAKNWLDKVTVRKMPHAITKDRRFTVDKFFFHVPITLNYKADSSPYRFNDFVRQYIKDCSDVKIIGIDRGERNLIYAVVIDGKGNIIEQRSFNTVGTYNYQEKLEQKEKERQTARQDWATVTKIKDLKKGYLSAVVHELSKMIVKYKAIVALENLNVGFKRMRGGIAERSVYQQFEKALIDKLNYLVFKDEEQSGYGGVLNAYQLTDKFESFSKMGQQTGFLFYVPAAYTSKIDPLTGFINPFSWKHVKNREDRRNFLNLFSKLYYDVNTHDFVLAYHHSNKDSKYTIKGNWEIADWDILIQENKEVFGKTGTPYCVGKRIVYMDDSTTGHNRMCAYYPHTELKKLLSEYGIEYTSGQDLLKIIQEFDDDKLVKGLFYIIKAALQMRNSNSETGEDYISSPIEGRPGICFDSRAEADTLPYDADANGAFHIAMKGLLLTERIRNDDKLAISNEEWLNYIQEMRG SEQ Cas12MNKDIRKNFTDFVGISEIQKTLRFILIPIGKTAQNIDKYNMFEDDEIRHEYY ID variantPILKEACDDFYRNHIDQQFENLELDWSKLDEALASEDRDLINETRATYRQ NO:VLFNRLKNSVDIKGDSKKNKTLSLESSDKNLGKKKTKNTFQYNFNDLFK 577AKLIKAILPLYIEYIYEGEKLENAKKALKMYNRFTSRLSNFWQARANIFTDDEISTGSPYRLVNDNFTIFRINNSIYTKNKPFIEEDILEFEKKLKSKKIIKDFESVDDYFTVNAFNKLCTQNGIDKYNSILGGFTTKEREKVKGLNELFNLAQQSINKGKKGEYRKNIRLGKLTKLKKQILAISDSTSFLIEQIEDDQDLYNKIKDFFELLLKEEIENENIFTQYANLQKLIEQADLSKIYINAKHLNKISHQVTGKWDSLNKGIALLLENININEESKEKSEVISNGQTKDISSEAYKRYLQIQSEEKDIERLRTQIYFSLEDLEKALDLVLIDENMDRSDKSILSYVQSPDLNVNFERDLTDLYSRIMKLEENNEKLLANHSAIDLIKEFLDLIMLRYSRWQILFCDSNYELDQTFYPIYDAVMEILSNIIRLYNLARNYLSRKPDRMKKKKINFNNPTLADGWSESKIPDNSSMLFIKDGMYYLGIIKNRAAYSELLEAESLQSSEKKKSENSSYERMNYHFLPDAFRSIPKSSIAMKAVKEHFEINQKTADLLLDTDKFSKPLRITKEIFDMQYVDLHKNKKKYQVDYLRDTGDKKGYRKALNTWLNFCKDFISKYKGRNLFDYSKIKDADHYETVNEFYNDVDKYSYHIFFTSVAETTVEKFISEGKLYLFQLYNKDFSPHSTGKPNLHTIYWRALFSEENLTSKNIKLNGQAEIFFRPKQIETPFTHKKGSILVNRFDVNGNPIPINVYQEIKGFKNNVIKWDDLNKTTQEGLENDQYLYFESEFEIIKDRRYTEDQLFFHVPISFNWDIGSNPKINDLATQYIVNSNDIHIIGIDRGENHLIYYSVIDLQGAIVEQGSLNTITEYTENKFLNNKTNNLRKIPYKDILQQREDERADARIKWHAIDKIKDLKDGYLGQIVHFLAKLIIKYNAIVILEDLNYGFKRGRFKVERQVYQKFEMALMKKLNVLVFKDYDIDEIGGPLKPWQLTRPIDSYERMGRQNGILFYVPAAYTSAVDPVTGFANLFYLNNVKNSEKFHFFSKFESIKYHSDQDMFSFAFDYNNFGTTTRINDLSKSKWQVFTNHERSVWNNKEKNYVTQNLTDLIKKLLRTYNIEFKNNQNVLDSILKIENNTDKENFARELFRLFRLTIQLRNTTVNENNTEITENELDYIISPVKDKNGNFFDSRDELKNLPDNGDANGAYNIARKGLLYIEQLQESIKTGKLPTLSISTLDWFNYIMK SEQ Cas12MTPIFCNFVVYQIMLFNNNININVKTMNKKHLSDFTNLFPVSKTLRFRLEP ID variantQGKTMENIVKAQTIETDEERSHDYEKTKEYIDDYHRQFIDDTLDKFAFKV NO:ESTGNNDSLQDYLDAYLSANDNRTKQTEEIQTNLRKAIVSAFKMQPQFNL 578LFKKEMVKHLLPQFVDTDDKKRIVAKFNDFTTYFTGFFTNRENMYSDEAKSTSIAYRIVNQNLIKFVENMLTFKSHILPILPQEQLATLYDDFKEYLNVASIAEMFELDHFSIVLTQRQIEVYNSVIGGRKDENNKQIKPGLNQYINQHNQAVKDKSARLPLLKPLFNQILSEKAGVSFLPKQFKSASEVVKSLNEAYAELSPVLAAIQDVVTNITDYDCNGIFIKNDLGLTDIAQRFYGNYDAVKRGLRNQYELETPMHNGQKAEKYEEQVAKHLKSIESVSLAQINQVVTDGGDICDYFKAFGATDDGDIQRENLLASINNAHTAISPVLNKENANDNELRKNTMLIKDLLDAIKRLQWFAKPLLGAGDETNKDQVFYGKFEPLYNQLDETISPLYDKVRSYLTKKPYSLDKFKINFEKSNLLGGWDPGADRKYQYNAVILRKDNDFYLGIMRDEATSKRKCIQVLDCNDEGLDENFEKVEYKQIKPSQNMPRCAFAKKECEENADIMELKRKKNAKSYNTNKDDKNALIRHYQRYLDRTYPEFGFVYKDADEYDTVKAFTDSMDSQDYKLSFLQVSETGLNKLVDEGDLYLFKITNKDFSSYAKGRPNLHTIYWRMLFDPKNLANVVYKLEGKAEVFFRRKSLASTTTHKAKQAIKNKSRYNEAVKPQSTFDYDIIKDRRFTADKFEFHVPIKMNFKAAGWNSTRLTNEVREFIKSQGVRHIIGIDRGERHLLYLTMIDMDGNIVKQCSLNAPAQDNARASEVDYHQLLDSKEADRLAARRNWGTIENIKELKQGYLSQVVHLLATMMVDNDAILVLENLNAGFMRGRQKVEKSVYQKFEKMLIDKLNYIVDKGQSPDKPTGALHAVQLTGLYSDFNKSNMKRANVRQCGFVFYIPAWNTSKIDPVTGFVNLFDTHLSSMGEIKAFFSKFDSIRYNQDKGWFEFKFDYSRFTTRAEGCRTQWTVCTYGERIWTHRSKNQNNQFVNDTVNVTQQMLQLLQDCGIDPNGNLKEAIANIDSKKSLETLLHLFKLTVQMRNSVTGSEVDYMISPVADERGHFFDSRESDEHLPANADANGAFNIARKGLMVVRQIMATDDVSKIKFAVSNKDWLRFAQHIDD SEQ Cas12MNKGGYVIMEKMTEKNRWENQFRITKTIKEEIIPTGYTKVNLQRVNMLK ID variantREMERNEDLKKMKEICDEYYRNMIDVSLRLEQVRTLGWESLIHKYRMLN NO:KDEKEIKALEKEQEDLRKKISKGFGEKKAWTGEQFIKKILPQYLMDHYTG 579EELEEKLRIVKKFKGCTMFLSTFFKNRENIFTDKPIHTAVGHRITSENAMLFAANINTYEKMESNVTLEIERLQREFWRRGINISEIFTDAYYVNVLTQKQIEAYNKICGDINQHMNEYCQKQKLKFSEFRMRELKKQILAVVGEHFEIPEKIESTKEVYRELNEYYESLKELHGQFEELKSVQLKYSQIYVQKKGYDRISRYIGGQWDLIQECMKKDCASGMKGTKKNHDAKIEEEVAKVKYQSIEHIQKLVCTYEEDRGHKVTDYVDEFIVSVCDLLGADHIITRDGERIELPLQYEPGTDLLKNDTINQRRLSDIKTILDWHMDMLEWLKTFLVNDLVIKDEEFYMAIEELNERMQCVISVYNRIRNYVTQKGYEPEKIRICFDKGTILTGWTTGDNYQYSGFLLMRNDKYYLGIINTNEKSVRKILDGNEECKDENDYIRVGYHLINDASKQLPRIFVMPKAGKKSEILMKDEQCDYIWDGYCHNKHNESKEFMRELIDYYKRSIMNYDKWEGYCFKFSSTESYDNMQDFYKEVREQSYNISFSYINENVLEQLDKDGKIYLFQVYNKDFAAGSTGTPNLHTMYLQNLFSSQNLELKRLRLGGNAELFYRPGTEKDVTHRKGSILVDRTYVREEKDGIEVRDTVPEKEYLEIYRYLNGKQKGDLSESAKQWLDKVHYREAPCDIIKDKRYAQEKYFLHFSVEINPNAEGQTALNDNVRRWLSEEEDIHVIGIDRGERNLIYVSLMDGKGRIKDQKSYNIVNSGNKEPVDYLAKLKVREKERDEARRNWKAIGKIKDIKTGYLSYVVHEIVEMAVREKAIIVMEDLNYGFKRGRFKVERQVYQKFEEMLINKLNYVVDKQLSVDEPGGLLRGYQLAFIPKDKKSSMRQNGIVFYVPAGYTSKIDPTTGFVNIFKFPQFGKGDDDGNGKDYDKIRAFFGKFDEIRYECDEKVTADNTREVKERYRFDFDYSKFETHLVHMKKTKWTVYAEGERIKRKKVGNYWTSEVISDIALRMSNTLNIAGIEYKDGHNLVNEICALRGKQAGIILNELLEIVRLTVQLRNSTTEGDVDERDEIISPVLNEKYGCFYHSTEYKQQNGDVLPKDADANGAYCIGLKGIYEIRQIKNKWKEDMTKGEGKALNEGMRISH DQWFEFIQNMNKGE SEQCas12 MNELVKNRCKQTKTICQKLIPIGKTRETIEKYNLMEIDRKIAANKELMNKL ID variantFSLIAGKHINDTLSKCTDLDFEPLLTSLSSLNNAKENDRDNLREYYDSVFE NO:EKKTLAEEISSRLTAVKFAGKDFFTKNIPDFLETYEGDDKNEMSELVSLVI 580ENTVTAGYVKKLEKIDRSMEYRLVSGTVVKRVLTDNADIYEKNIEKAKDFDYGVLNIDEASQFTTLVAKDYANYLTADGIAIYNVGIGKINLALNEYCQKNKEYSYNKLALLPLQKMLYGEKLSLFEKLEDFTSDEELINSYNKFAKTVNESGLAEIIKKAVPSYDEIVIKPNKISNYSNSITGHWSLVNRIMKDYLENNGIKNADKYMEKGLTLSEIGDALENKNIKHSDFISNLINDLGHTYTEIKENKESLKKDESVNALIIKKELDMLLSILQNLKVFDIDNEMFDTGFGIEVSKAIEILGYGVPLYNKIRNYITKKPDPKKKFMTKFGSATIGTGITTSVEGSKKATFLKDGDAVFLLLYNTAGCKANNVSVSNLADLINSSLEIENSGKCYQKMIYQTPGDIKKQIPRVFVYKSEDDDLIKDFKAGLHKTDLSFLNGRLIPYLKEAFATHETYKNYTFSYRNSYESYDEFCEHMSEQAYILEWKWIDKKLIDDLVEDGSLLMFRVWNRFMKKKEGKISKHAKIVNELFSDENASNAAIKLLSVFDIFYRDKQIDNPIVHKAGTTLYNKRTKDGEVIVDYTTMVKNKEKRPNVYTTTKKYDIIKDRRYTEEQFEIHLHVNIGKEENKEKLETSKVINEKKNTLVVTRSNEHLLYVVIFDENDNILLKKSLNTVKGMNFKSKLEVVEIQKKENMQSWKTVGSNQALMEGYLSFAIKEIADLVKEYDAILVLEQNSVGKNILNERVYTRFKEMLITNLSLDVDYENKDFYSYTELGGKVASWRDCVTNGICIQVPSAYKYKDPTTSFSTISMYAKTTAEKSKKLKQIKSFKYNRERGLFELVIAKGVGLENNIVCDSFGSRSIIENDISKEVSCTLKIEKYLIDAGIEYNDEKEVLKDLDTAAKTDAVHKAVTLLLKCFNESPDGRYYISPCGEHFTLCDAPEVLSAINYYIRSRYIRE QIVEGVKKMEYKKTILLAKSEQ Cas12 MNYKTGLEDFIGKESLSKTLRNALIPTESTKIHMEEMGVIRDDELRAEKQQ ID variantELKEIMDDYYRTFIEEKLGQIQGIQWNSLFQKMEETMEDISVRKDLDKIQN NO:EKRKEICCYFTSDKRFKDLFNAKLITDILPNFIKDNKEYTEEEKAEKEQTRV 581LFQRFATAFTNYFNQRRNNFSEDNISTAISFRIVNENSEIHLQNMRAFQRIEQQYPEEVCGMEEEYKDMLQEWQMKHIYSVDFYDRELTQPGIEYYNGICGKINEHMNQFCQKNRINKNDFRMKKLHKQILCKKSSYYEIPFRFESDQEVYDALNEFIKTMKKKEIIRRCVHLGQECDDYDLGKIYISSNKYEQISNALYGSWDTIRKCIKEEYMDALPGKGEKKEEKAEAAAKKEEYRSIADIDKIISLYGSEMDRTISAKKCITEICDMAGQISIDPLVCNSDIKLLQNKEKTTEIKTILDSFLHVYQWGQTFIVSDIIEKDSYFYSELEDVLEDFEGITTLYNHVRSYVTQKPYSTVKFKLHFGSPTLANGWSQSKEYDNNAILLMRDQKFYLGIFNVRNKPDKQIIKGHEKEEKGDYKKMIYNLLPGPSKMLPKVFITSRSGQETYKPSKHILDGYNEKRHIKSSPKFDLGYCWDLIDYYKECIHKHPDWKNYDFHFSDTKDYEDISGFYREVEMQGYQIKWTYISADEIQKLDEKGQIFLFQIYNKDFSVHSTGKDNLHTMYLKNLFSEENLKDIVLKLNGEAELFFRKASIKTPIVHKKGSVLVNRSYTQTVGNKEIRVSIPEEYYTEIYNYLNHIGKGKLSSEAQRYLDEGKIKSFTATKDIVKNYRYCCDHYFLHLPITINFKAKSDVAVNERTLAYIAKKEDIHIIGIDRGERNLLYISVVDVHGNIREQRSFNIVNGYDYQQKLKDREKSRDAARKNWEEIEKIKELKEGYLSMVIHYIAQLVVKYNAVVAMEDLNYGFKTGRFKVERQVYQKFETMLIEKLHYLVFKDREVCEEGGVLRGYQLTYIPESLKKVGKQCGFIFYVPAGYTSKIDPTTGFVNLFSFKNLTNRESRQDFVGKFDEIRYDRDKKMFEFSFDYNNYIKKGTILASTKWKVYTNGTRLKKIVVNGKYTSQSMEVELTDAMEKMLQRAGIEYHDGKDLKGQIVEKGIEAEIIDIFRLTVQMRNSRSESEDREYDRLISPVLNDKGEFFDTATADKTLPQDADANGAYCIALKGLYEVKQIKENWKENEQFPRNKLVQDNKTWFDFMQKKRYL SEQ Cas12MEDKQFLERYKEFIGLNSLSKTLRNSLIPVGSTLKHIQEYGILEEDSLRAQK ID variantREELKGIMDDYYRNYIEMHLRDVHDIDWNELFEALTEVKKNQTDDAKKR NO:LEKIQEKKRKEIYQYLSDDAVFSEMFKEKMISGILPDFIRCNEGYSEEEKEE 582KLKTVALFHRFTSSFNDFFLNRKNVFTKEAIVTAIGYRVVHENAEIFLENMVAFQNIQKSAESQISIIERKNEHYFMEWKLSHIFTADYYMMLMTQKAIEHYNEMCGVVNQQMREYCQKEKKNWNLYRMKRLHKQILSNASTSFKIPEKYENDAEVYESVNSFLQNVMEKTVMERIAVLKNSTDNFDLSKIYITAPYYEKISNYLCGSWNTITDCLTHYYEQQIAGKGARKDQKVKAAVKADKWKSLSEIEQLLKEYARAEEVKRKPEEYIAEIENIVSLKEAHLLEYHPEVNLIENEKYATEIKDVLDNYMELFHWMKWFYIEEAVEKEVNFYGELDDLYEEIKDIVPLYNKVRNYVTQKPYSDTKIKLNFGTPTLANGWSKSKEYDYNAILLQKDGKYYMGIFNPIQKPEKEIIEGHSQPLEGNEYKKMVYYYLPSANKMLPKVLLSKKGMEIYQPSEYIINGYKERRHIKSEEKFDLQFCHDLIDYFKSGIERNSDWKVFGFDFSDTDTYQDISGFYREVEDQGYKIDWTYIKEADIDRLNEEGKLYLFQIYNKDFSEKSTGRENLHTMYLKNLFSEENVREQVLKLNGEAEIFFRKSSVKKPIIHKKGTMLVNRTYMEEVNGNSVRRNIPEKEYQEIYNYKNHRLKGELSTEAKKYLEKAVCHETKKDIVKDYRYSVDKFFIHLPITINYRASGKETLNSVAQRYIAHQNDMHVIGIDRGERNLIYVSVINMQGEIKEQKSFNIINEFNYKEKLKEREQSRGAARRNWKEIGQIKDLKEGYLSGVIHEIAKMMIKYHAIIAMEDLNYGFKRGRFKVERQVYQKFENMLIQKLNYLVFKDRPADEDGGVLRGYQLAYIPDSVKKMGRQCGMIFYVPAAFTSKIDPTTGFVDIFKHKVYTTEQAKREFILSFDEICYDVERQLFRFTFDYANFVTQNVTLARNNWTIYTNGTRAQKEFGNGRMRDKEDYNPKDKMVELLESEGIEFKSGKNLLPALKKVSNAKVFEELQKIVRFTVQLRNSKSEENDVDYDHVISPVLNEEGNFFDSSKYKNKEEKKESLLPVDADANGAYCIALKGLYIMQAIQKNWSEEKALSPDVL RLNNNDWFDYIQNKRYR SEQCas12 MEKSLNDFIGLYSVSKTLRFELKPVSETLENIKKFHFLEEDKKKANDYKD ID variantVKKIIDNYHKYFIDDVLKNASFNWKKLEEAIREYNKNKSDDSALVAEQK NO:KLGDAILKLFTSDKRYKALTAATPKELFESILPDWFGEQCNQDLNKAALK 583TFQKFTSYFTGFQENRKNVYSAEAIPTAVPYRIVNDNFPKFLQNVLIFKTIQEKCPQIIDEVEKELSSYLGKEKLAGIFTLESFNKYLGQGGKENQRGIDFYNQIIGGVVEKEGGINLRGVNQFLNLYWQQHPDFTKEDRRIKMVPLYKQILSDRSSLSFKIESIENDEELKNALLECADKLELKNDEKKSIFEEVCDLFSSVKNLDLSGIYINRKDINSVSRILTGDWSWLQSRMNVYAEEKFTTKAEKARWQKSLDDEGENKSKGFYSLTDLNEVLEYSSENVAETDIRITDYFEHRCRYYVDKETEMFVQGSELVALSLQEMCDDILKKRKAMNTVLENLSSENKLREKTDDVAVIKEYLDAVQELLHRIKPLKVNGVGDSTFYSVYDSIYSALSEVISVYNKTRNYITKKAASPEKYKLNFDNPTLADGWDLNKEQANTSVILRKDGMFYLGIMNPKNKPKFAEKYDCGNESCYEKMIYKQFDATKQIPKCSTQKKEVQKYFLSGATEPYILNDKKSFKSELIITKDIWFMNNHVWDGEKFVPKRDNETRPKKFQIGYFKQTGDFDGYKNALSNWISFCKNFLQSYLSATVYDYNFKNSEEYEGLDEFYNYLNATCYKLNFINIPETEINKMVSEGKLYLFQIYNKDFASGSTGMPNMHTLYWKNLFSDENLKNVCLKLNGEAELFYRPAGIKEPVIHKEGSYLVNRTTEDGESIPEKIYFEIYKNANGKLEKLSDEAQNYISNHEVVIKKAGHEIIKDRHYTEPKFLFHVPLTINFKASGNSYSINENVRKFLKNNPDVNIIGLDRGERHLIYLSLINQKGEIIKQFTFNEVERNKNGRTIKVNYHEKLDQREKERDAARKSWQAIGKIAELKEGYLSAVIHQLTKLMVEYNAVVVMEDLNFGFKRGRFHVEKQVYQKFEHILIDKSNYLVFKDRGLNEPGGVLNGYQIAGQFESFQKLGKQSGMLFYVPAGYTSKIDPKTGFVSMMNFKDLTNVHKKRDFFSKFDNIHYDEANGSFVFTFDYKKFDGKAKEEMKLTKWSVYSRDKRIVYFAKTKSYEDVLPTEKLQKIFESNGIDYKSGNNIQDSVMAIGADLKEGAKPSKEISDFWDGLLSNFKLILQMRNSNARTGEDYIISPVMADDGTFFDSREEFKKGEDAKLPLDADANGAYHIALKGLSLINKINLSKDEELKKFDMKISNADW FKFAQEKNYAK SEQ Cas12MEEKKMSKIEKFIGKYKISKTLRFRAVPVGKTQDNIEKKGILEKDKKRSED ID variantYEKVKAYLDSLHRDFIENTLKKVKLNELNEYACLFFSGTKDDGDKKKME NO:KLEEKMRKTISNEFCNDEMYKKIFSEKILSENNEEDVSDIVSSYKGFFTSLN 584GYVNNRKNLYVSDAKPTSIAYRCINENLPKFLRNVECYKKVVQVIPKEQIEYMSNNLNLSPYRIEDCFNIDFFEFCLSQGGIDLYNTFIGGYSKKDGTKVQGINEIVNLYNQKNKKDKEKYKLPQFTPLFKQILSDRDTKSFSIEKLENIYEVVELVKKSYSDEMFDDIETVFSNLNYYDASGIYVKNGPAITHISMNLTKDWATIRNNWNYEYDEKHSTKKNKNIEKYEDTRNTMYKKIDSFTLEYISRLVGKDIDELVKYFENEVANFVMDIKKTYSKLTPLFDRCQKENFDISEDEVNDIKGYLDNVKLLESFMKSFTINGKENNIDYVFYGKFTDDYDKLHEFDHIYNKVRNYITTSRKPYKLDKYKLYFDNPQLLGGWDINKEKDYRTVMLTKDGKYYFAIIDKGEHPFDNIPKDYFDNNGYYKKIIYRQIPNAAKYLSSKQIVPQNPPEEVKRILDKKKADSKSLTEEEKNIFIDYIKSDFLKNYKLLFDKNNNPYFNFAFRESSTYESLNEFFEDVERQAYSVRYENLPADYIDNLVNEGKIYLFEIYSKDFSEYSKGTNNLHTMYFKALFDNDNLKNTVFKLSGNAELFIRPASIKKDELVIHPKNQLLQNKNPLNPKKQSIFDYDLVKDKRFFENQYMLHISIEINKNERDAKKIKNINEMVRKELKDSDDNYIIGIDRGERNLLYVCVINSAGKIVEQMSLNEIINEYNGIKHTVDYQGLLDKCEKERNAQRQSWKSIENIKELKDGYISQVVHKLCQLVEKYDAIIAMENLNGGFKRGRTKFEKQVYQKFENKLINKMEYMADKKRKTTENGGILRGYQLTNGCINNSYQNGFIFYVPAWLTSKIDPTTGFVDLLKPKYTNVEEAHLWINKFNSITYDKKLDMFAFNINYSQFPRADIDYRKIWTFYTNGYRIETFRNSEKNNEFDWKEVHLTSVIKKLLEEYQINYISGKNIIDDLIQIKDKPFWNSFIKYIRLTLQMRNSITGRTDVDYIISPVINNEGTFYDSRKDLDEITLPQDADANGAYNIARKALWIIEKLKESPDEELNKVKLAI TQREWLEYAQINI SEQCas12 MIIHNCYIGGSFMKKIDSFTNCYSLSKTLRFKLIPIGATQSNFDLNKMLDED ID variantKKRAENYSKAKSIIDKYHRFFIDKVLSSVTENKAFDSFLEDVRAYAELYY NO:RSNKDDSDKASMKTLESKMRKFIALALQSDEGFKDLFGQNLIKKTLPEFL 585ESDTDKEIIAEFDGFSTYFTGFFNNRKNMYSADDQPTAISYRCINDNLPKFLDNVRTFKNSDVASILNDNLKILNEDFDGIYGTSAEDVFNVDYFPFVLSQKGIEAYNSILGGYTNSDGSKIKGLNEYINLYNQKNENIHRIPKMKQLFKQILSERESVSFIPEKFDSDDDVLSSINDYYLERDGGKVLSIEKTVEKIEKLFSAVTDYSTDGIFVKNAAELTAVCSGAFGYWGTVQNAWNNEYDALNGYKETEKYIDKRKKAYKSIESFSLADIQKYADVSESSETNAEVTEWLRNEIKEKCNLAVQGYESSKDLISKPYTESKKLFNNDNAVELIKNALDSVKELENVLRLLLGTGKEESKDENFYGEFLPCYERICEVDSLYDKVRNYMTQKLYKTDKIKLNFQNPQFLGGWDRNKEADYSAVLLRRNSLYYIAIMPSGYKRVFEKIPAPKADETVYEKVIYKLLPGPNKMLPKVFFSKKGIETFNPPKEILEKYELGTHKTGDGFNLDDCHALIDYFKSALDVHSDWSNFGFRFSDTSTYKNIADFYNEVKNQGYKITFCDVPQSYINELVDEGKLYLFQLYNKDFSEHSKGTPNLHTLYFKMLFDERNLENVVFKLNGEAEMFYREASISKDDMIVHPKNQPIKNKNEQNSRKQSTFEYDIVKDRRYTVDQFMLHIPITLNFTANGGTNINNEVRKALKDCDKNYVIGIDRGERNLLYICVVDSEGRIIEQYSLNEIINEYNGNTYSTDYHALLDKKEKERLESRKAWKTVENIKELKEGYISQVVHKICELVEKYDAVIVMEDLNLGFKQGRSGKFEKSVYQKFEKMLIDKLNYFADKKKSPEEIGSVLNAYQLTNAFESFEKMGKQNGFIFYVPAYLTSKIDPTTGFADLLHPSSKQSKESMRDFVGRFDSITFNKTENYFEFELDYNKFPRCNTDYRKKWTVCTYGSRIKTFRNPEKNSEWDNKTVELTPAFMALFEKYSIDVNGDIKAQIMSVDKKDFFVELIGLLRLTLQMRNSETGKVDRDYLISPVKNSEGVFYNSDDYKGIENASLPKDADANGAYNIARKGLWIIEQIKACENDAELNKIRLAISNAEWLEYAQKK SEQ Cas12MKEQFVNQYPISKTLRFSLIPIGKTEENFNKNLLLKEDEKKAEEYQKVKGY ID variantIDRYHKFFIETALCNINFEGFEEYSLLYYKCSKDDNDLKTMEDIEIKLRKQI NO:SKTMTSHKLYKDLFGENMIKTILPNFLDSDEEKNSLEMFRGFYTYFSGFNT 586NRKNMYTEEAKSTSIAYRCINDNLPKFLDNSKSFEKIKCALNKEELKAKNEEFYEIFQIYATDIFNIDFFNFVLTQPGIDKYNGIIGGYTCSDGTKVQGLNEIINLYNQQIAKDDKSKRLPLLKMLYKQILSDRETVSFIPEKFSSDNEVLESINNYFSKNVSNAIKSLKELFQGFEAYNMNGIFISSGVAITDLSNAVFGDWNAISTAWEKAYFETNPPKKNKSQEKYEEELKANYKKIKSFSLDEIQRLGSIAKSPDSIGSVAEYYKITVTEKIDNITELYDGSKELLNCNYSESYDKKLIKNDTVIEKVKTLLDAVKSLEKLIKPLVGTGKEDKDELFYGTFLPLYTSLSAVDRLYDKVRNYATQKPYSKDKIKLNFNCSSFLSGWATDYSSNGGLIFEKDGLYYLGIVNKKFTTEEIDYLQQNADENPAQRIVYDFQKPDNKNTPRLFIRSKGTNYSPSVKEYNLPVEEIVELYDKRYFTTEYRNKNPELYKASLVKLIDYFKLGFTRHESYRHYDFKWKKSEEYNDISEFYKDVEISCYSLKQEKINYNTLLNFVAENRIYLFQIYNKDFSKYSKGTPNLHTRYFKALFDENNLSDVVFKLNGGSEMFFRKASIKDNEKVVHPANQPIDNKNPDNSKKQSTFDYELIKDKRFTKHQFSIHIPITMNFKARGRDFINNDIRKAIKSEYKPYVIGIDRGERNLIYISVINNNGEIVEQMSLNDIISDNGYKVDYQRLLDRKEKERDNARKSWGTIENIKELKEGYISQVIHKICELVIKYDAVIAMEDLNFGFKRGRFNVEKQVYQKFENMLISKLNYLCDKKSEANSEGGLLKAYQLTNKFDGVNKGKQNGIIFYVPAWLTSKIDPVTGFVDLLHPKYISVEETHSLFEKLDDIRYNFEKDMFEFDIDYSKLPKCNADFKQKWTVCTNADRIMTFRNSEKNNEWDNKRILLSDEFKRLFEEFGIDYCHNLKNKILSISNKDFCYRFIKLFALTMQMRNSITGSTNPEDDYLISPVRDENGVFYDSRNFIGSKAGLPIDADANGAYNIARKGLWAINAIKSTADD MLDKVDLSISNAKWLEYVQKSEQ Cas12 MADLSQFTHKYQVPKTLRFELIPQGKTLENLSAYGMVADDKQRSENYKK ID variantLKPVIDRIYKYFIEESLKNTNLDWNPLYEAIREYRKEKTTATITNLKEQQDI NO:CRRAIASRFEGKVPDKGDKSVKDFNKKQSKLFKELFGKELFTDSVLEQLP 587GVSLSDEDKALLKSFDKFTTYFVGFYDNRKNVFSSDDISTGIPHRLVQENFPKFIDNCDDYKRLVLVAPELKEKLEKAAEATKIFEDVSLDEIFSIKFYNRLLQQNQIDQFNQLLGGIAGAPGTPKIQGLNETLNLSMQQDKTLEQKLKSVPHRFSPLYKQILSDRSSLSFIPESFSCDAEVLLAVQEYLDNLKTEHVIEDLKEVFNRLTTLDLKHIYVNSTKVTAFSQALFGDWNLCREQLRVYKMSNGNEKITKKALGELESWLKNSDIAFTELQEALADEALPAKVNLKVQEAISGLNEQMAKSLPKELKIPEEKEELKALLDAIQEVYHTLEWFIVSDDVETDTDFYVPLKETLQIIQPIIPLYNKVRNFATQKPYSVEKFKLNFANPTLADGWDENKEQQNCAVLFQKGNNYYLGILNPKNKPDFDNVDTEKQGNCYQKMVYKQFPDFSKMMPKCTTQLKEVKQHFEGKDSDYILNNKNFIKPLTITREVYDLNNVLYDGKKKFQIDYLRKTKDEDGYYTALHTWIDFAKKFVASYKSTSIYDTSTILPPEKYEKLNEFYGALDNLFYQIKFENIPEEIIDTYVEDGKLFLFQIYNKDFAAGATGAPNLHTIYWKAVFDPENVKDVVVKLNGQAELFYRPKSNMDVIRHKVGEKLVNRTLKDGSILTDELHKELYLYANGSLKKGLSEDAKIILDKNLAVIYDVHHEIVKDRRFTTDKFFFHVPLTLNYKCDKNPVKFNAEVQEYLKENPDTYVIGIDRGERNLIYAVVIDPKGRIVEQKSFNVINGFDYHGKLDQREKERVKARQAWTAVGKIKELKQGYLSLVVHEISKMMVRYQAVVVLENLNVGFKRVRSGIAEKAVYQQFEKMLINKLNYLMFKDAGGTEPGSVLNAYQLTDRFESFAKMGLQTGFLFYIPAAFTSKIDPATGFVDPFRWGAIKTLADKREFLSGFESLKFDSTTGNFILHFDVSKNKNFQKKLEGFVPDWDIIIEANKMKTGKGATYIAGKRIEFVRDNNSQGHYEDYLPCNALAETLRQCDIPYEEGKDILPLILEKNDSKLLHSVFKVVRLTLQMRNSNAETGEDYISSPVEDVSGSCFDSRMENEKLPKDADANGAYHIALKGMLALERLRKDEKMAISNNDWLNYIQEKR A SEQ Cas12MTNFDNFTKKYVNSKTIRLEAIPVGKTLKNIEKMGFIAADRQRDEDYQKA ID variantKSVIDHIYKAFMDDCLKDLFLDWDPLYEAVVACWRERSPEGRQALQIMQ NO:ADYRKKIADRFRNHELYGSLFTKKIFDGSVAQRLPDLEQSAEEKSLLSNFN 588KFTSYFRDFFDKRKRLFSDDEKHSAIAYRLINENFLKFVANCEAFRRMTERVPELREKLQNTGSLQVYNGLALDEVFSADFYNQLIVQKQIDLYNQLIGGIAGEPGTPNIQGLNATINLALQGDSSLHEKLAGIPHRFNPLYKQILSDVSTLSFVPSAFQSDGEMLAAVRGFKVQLESGRVLQNVRRLFNGLETEADLSRVYVNNSKLAAFSSMFFGRWNLCSDALFAWKKGKQKKITNKKLTEIKKWLKNSDIAIAEIQEAFGEDFPRGKINEKIQAQADALHSQLALPIPENLKALCAKDGLKSMLDTVLGLYRMLQWFIVGDDNEKDSDFYFGLGKILGSLDPVLVLYNRVRNYITKKPYSLTKFRLNFDNSQLLNGWDENNLDTNCASIFIKDGKYYLGISNKNNRPQFDTVATSGKSGYQRMVYKQFANWGRDLPHSTTQMKKVKKHFSASDADYVLDGDKFIRPLIITKEIFDLNNVKFNGKKKLQVDYLRNTGDREGYTHALHTWINFAKDFCACYKSTSIYDISCLRPTDQYDNLMDFYADLGNLSHRIVWQTIPEEAIDNYVEQGQLFLFQLYNKDFAPGADGKPNLHTLYWKAVFNPENLEDVVVKLNGKAELFYRPRSNMDVVRHKVGEKLVNRKLKNGLTLPSRLHEEIYRYVNGTLNKDLSADARSVLPLAVVRDVQHEIIKDRRFTADKFFFHASLTFNFKSSDKPVGFNEDVREYLREHPDTYVVGVDRGERNLIYIVVIDPQGNIVEQRSFNMINGIDYWSLLDQKEKERVEAKQAWETVGKIKDLKCGYLSFLIHEITKIIIKYHAVVILENLSLGFKRVRTGIAEKAVYQQFERMLVTKLGYVVFKDRAGKAPGGVLNAYQLTDNTRTAENTGIQNGFLFYVPAAFTSRVDPATGFFDFYDWGKIKTATDKKNFIAGFNSVRYERSTGDFIVHVGAKNLAVRRVAEDVRTEWDIVIEANVRKMGIDGNSYISGKRIRYRSGEQGHGQYENHLPCQELIRALQQYGIQYETGKDILPAILQQDDAKLTDTVFDVFRLALQMRNTSAETGEDYFNSVVRDRSGRCFDTRRAEAAMPKEADANDAYHIALKGLFVLEKLRKGESIGIKNTEWLRYVQQRHS SEQ Cas12MENYGGFTGLYPLQKTLKFELRPQGRTMEHLVSSNFFEEDRDRAEKYKIV ID variantKKVIDNYHKDFINECLSKRSFDWTPLMKTSEKYYASKEKNGKKKQDLDQ NO:KIIPTIENLSEKDRKELELEQKRMRKEIVSVFKEDKRFKYLFSEKLFSILLKD 589EDYSKEKLTEKEILALKSFNKFSGYFIGLHKNRANFYSEGDESTAIAYRIVNENFPKFLSNLKKYREVCEKYPEIIQDAEQSLAGLNIKMDDIFPMENFNKVMTQDGIDLYNLAIGGKAQALGEKQKGLNEFLNEVNQSYKKGNDRIRMTPLFKQILSERTSYSYILDAFDDNSQLITSINGFFTEVEKDKEGNTFDRAVGLIASYMKYDLSRVYIRKADLNKVSMEIFGSWERLGGLLRIFKSELYGDVNAEKTSKKVDKWLNSGEFSLSDVINAIAGSKSAETFDEYILKMRVARGEIDNALEKIKCINGNFSEDENSKMIIKAILDSVQRLFHLFSSFQVRADFSQDGDFYAEYNEIYEKLFAIVPLYNRVRNYLTKNNLSMKKIKLNFKNPALANGWDLNKEYDNTAVIFLREGKYYLGIMNPSKKKNIKFEEGSGTGPFYKKMAYKLLPDPNKMLPKVFFAKKNINYYNPSDEIVKGYKAGKYKKGENFDIDFCHKLIDFFKESIQKNEDWRAFNYLFSATESYKDISDFYSEVEDQGYRMYFLNVPVANIDEYVEKGDLFLFQIYNKDFASGAKGNKDMHTIYWNAAFSDENLRNVVVKLNGEAELFYRDKSIIEPICHKKGEMLVNRTCFDKTPVPDKIHKELFDYHNGRAKTLSIEAKGYLDRVGVFQASYEIIKDRRYSENKMYFHVPLKLNFKADGKKNLNKMVIEKFLSDKDVHIIGIDRGERNLLYYSVIDRRGNIIDQDSLNIIDGFDYQKKLGQREIERREARQSWNSIGKIKDLKEGYLSKAVHKVSKMVLEYNAIVVLEDLNFGFKRGRFKVEKQVYQKFEKMLIDKLNYLVFKEVLDSRDAGGVLNAYQLTTQLESFNKLGKQSGILFYVPAAYTSKIDPTTGFVSLFNTSRIESDSEKKDFLSGFDSIVYSAKDGGIFAFKFDYRNRNFQREKTDHKNIWTVYTNGDRIKYKGRMKGYEITSPTKRIKDVLSSSGIRYDDGQELRDSIIQSGNKVLINEVYNSFIDTLQMRNSDGEQDYIISPVKNRNGEFFRTDPDRRELPVDADANGAYHIALRGELLMQKIAEDFDPKSDKFTMPKMEHKDWFEFM QTRGD SEQ Cas12MLHAFTNQYQLSKTLRFGATLKEDEKKCKSHEELKGFVDISYENMKSSA ID variantTIAESLNENELVKKCERCYSEIVKFHNAWEKIYYRTDQIAVYKDFYRQLS NO:RKARFDAGKQNSQLITLASLCGMYQGAKLSRYITNYWKDNITRQKSFLK 590DFSQQLHQYTRALEKSDKAHTKPNLINFNKTFMVLANLVNEIVIPLSNGAISFPNISKLEDGEESHLIEFALNDYSQLSELIGELKDAIATNGGYTPFAKVTLNHYTAEQKPHVFKNDIDAKIRELKLIGLVETLKGKSSEQIEEYFSNLDKFSTYNDRNQSVIVRTQCFKYKPIPFLVKHQLAKYISEPNGWDEDAVAKVLDAVGAIRSPAHDYANNQEGFDLNHYPIKVAFDYAWEQLANSLYTTVTFPQEMCEKYLNSIYGCEVSKEPVFKFYADLLYIRKNLAVLEHKNNLPSNQEEFICKINNTFENIVLPYKISQFETYKKDILAWINDGHDHKKYTDAKQQLGFIRGGLKGRIKAEEVSQKDKYGKIKSYYENPYTKLTNEFKQISSTYGKTFAELRDKFKEKNEITKITHFGIIIEDKNRDRYLLASELKHEQINHVSTILNKLDKSSEFITYQVKSLTSKTLIKLIKNHTTKKGAISPYADFHTSKTGFNKNEIEKNWDNYKREQVLVEYVKDCLTDSTMAKNQNWAEFGWNFEKCNSYEDIEHEIDQKSYLLQSDTISKQSIASLVEGGCLLLPIINQDITSKERKDKNQFSKDWNHIFEGSKEFRLHPEFAVSYRTPIEGYPVQKRYGRLQFVCAFNAHIVPQNGEFINLKKQIENFNDEDVQKRNVTEFNKKVNHALSDKEYVVIGIDRGLKQLATLCVLDKRGKILGDFEIYKKEFVRAEKRSESHWEHTQAETRHILDLSNLRVETTIEGKKVLVDQSLTLVKKNRDTPDEEATEENKQKIKLKQLSYIRKLQHKMQTNEQDVLDLINNEPSDEEFKKRIEGLISSFGEGQKYADLPINTMREMISDLQGVIARGNNQTEKNKIIELDAADNLKQGIVANMIGIVNYIFAKYSYKAYISLEDLSRAYGGAKSGYDGRYLPSTSQDEDVDFKEQQNQMLAGLGTYQFFEMQLLKKLQKIQSDNTVLRFVPAFRSADNYRNILRLEETKYKSKPFGVVHFIDPKFTSKKCPVCSKTNVYRDKDDILVCKECGFRSDSQLKERENNIHYIHN GDDNGAYHIALKSVENLIQMKSEQ Cas12 MKNGINLFKTKTTKTKGVDMEKYQITKTIRFKLLPDNAHEIVEKVKSLKT ID variantSNVDELMDEVKNVHLKGLELLFALKKYFYFDGNQCKSFKSTLEIKARWL NO:RLYTPDQYYLKKSSKNSYQLKSLSYFKDVFNDWLFNWEESVSELAIIYEK 591YKICQHQRDSRADIALLIKKLSMKEYFPFISDLIDCVNDKNSNKTFLMKLSEELSVLLEKCNSRALPYQSNGIVVGKASLNYYTVSKSEKMLQNEYEDVCQSLDKNYDITEMKVILYKEKLDNLNFKDVTIANAYNLLKENKALQKRLFSEYVSQGKVLSLIKTELPLFSNINDNDFEKYKEWSNEIKKLADKKNTFCKKTQQDKIKDIQNKISELKKKRGALFQYKFTSFQKHCDNYKKVAVQYGKLKARKKAIEKDEIEANLLRYWSVILEQEDKHSLVLIPKNNAKDAKQYIETINTKGGKYIIHEILDSLTLRALNKLCFNAVDIEKGQMVRENTFYQGIKEEFERNKINCDNQGVLKIQGLYSFKTEGGQINEKEAVEFFKEVLKSNYAREVLNLPYDLESNIFQKEYTNLDQFRQDLEKCCYALHSKIGKDDLDEFTRRFEAQVFDITSIDLKSKKEKTKTTGEMKKHTQLWLEFWKGAIEQNFATRVNPELSIFWRAPKSSREKKYGKGSDLYDPNKNNRYLYEQYTLALTITENAGSHFKDIAFKDTSKIKEAIKEFNMSLSQSKYCFGIDRGNAELVSLCLIKNEKDFPFEKFPVYRLRDLTYQGDFKDKHDQMRYGVAIKNISYFIDQEDLFEKNNLSAIDMTTAKLIKNKIVLNGDVLTYLKLKEETAKHKLTQFFQGSSINKNSRVYFDEDENVFKITTNRNHNPEEIIYFYRGEYGAIKNKNDLEDILNEYLCKMETGESEIVLLNRVNHLRDAISANIVGILSYLIDLFPETIVALENLAKGTIDRHVSQSYENITRRFEWALYRKLLNKQLAPPELKENILLREGDDKIDQFGIIHFVEEKNTSKDCPNCRKTTQQTNDNKFKEKKFVCKSCGFDTSKDRKGMDSLNSPDTVAAYN VARKKFES SEQ Cas12MAKETKEFKTFDDFTNLYEVQKTLRFELEAVPETEIVLENRGIWYKRDKK ID variantRADEKPIVKFYMDILHREFTDEALEKIKESGVLNLSGYFKLFEELRRLQNH NO:GANTKEEKKLKLEEIRAKKREISNELSQIRRVFSVRGFDVVDSDWKKKYTI 592EGKKIKNDKSKTYLILSENILNFLENRFTSKEVERLRSIDKKHVEDYGNVVNSGGENIFATFKGFFGYFDSLIKNRENFYETDGKAGRVATRSVDENLNFFAENLHIFSTDLPKALKDDLSDTQKAIFERSYYKNCLLQKDIKSYNLIIGDINKEINKHRQQRDTKIKFLNTLFKQILSIEEKEQYKHIEINNDEDLIRAIRDFISLNESKISEGTKIFNQFIQRCLQKEDLGQIYLPKDSVNTIAHRIFKPWDEIMALFDRKYFVSLEEIKDLTESSVWKERVLEESKTKSLIFKDTHIHTIISGQEIFSNFILILEKEYKNQFSGFISETRRGKAAFVGYDESLKNLRATIKWFEGKNLKLSETEKVEWIKAIKDYADAALRIFQMTKYLWLPVVGDEEDKDYLRIKAEIDQLTKDNDFYNKINAFIDGYKPEPFIYRSSFQEYLTRRPFSTDKFKINFENSRLLDGWDKDMIDDRMGILLQRDGDYFLGILNKEDRHCLDNLVDVKSEDKNSYALMQFKQLTGLYRQLPRMAFPKKKQPVLEANAEIKKIKEDFDFLQKQKKEREVNVNVVFDNKKLNLLINHYAEFLKENYKDEKCYDFSLLNKEKVYESLSDFYADVDKITYSLSFIQVSIDQLIKTGKILLFRLKNKDLLKGSLGQNKNLHTYYFHALFERENLSQGRIRLGAQAEIFFRPASIEKEKDKNRSNALKKSPKTRYVKEILKNKRYSEDKVFLHLPIQLNADAYDLPSINQNVFEFIKNRQEKVKIIGIDRGEKNLAYYSVISQNSNGKIKIEEPPRDLNLGYLEPLDELENKRQDERKAWQSISEIKSKRDGYISYAVSKIVELMLKYQAIIVLEDLSGKFKRSRMKFEKAPYQQLELALIKKLNYLVKKNSKSGKPGHYLSAYQLTEPVGSYKEMGKQTGIIFYTQAGYTSRTCPTCGWRKRVQGLYYKDRTSAQRRFDPKTGVKIFYDSVNDRFVFQYHPVYEQKELKEWDKEIYSDVTRIRWNNEEKKNNEYRKGDITLKIKRLFRDRGIDLSRNINEQLVNVGDASFWEELINLLRLITEIRNIDNENNRDFIECPHCHFQSENGFHGVAWNGDANGAYNIARKGLLITKAVCDPEKNVGDITWSDLKVDMKDWDAATDEWAKKNPEK SEQ Cas12MENEKIFSDLTNRYQVVKTLPFELKPVPRTRVLLGLDNPNKGEIFSKDRER ID variantAENFTIIKKYIDRLHSLFINESLKKADIDFSNFYKQYGKNINTKNNKNIDDD NO:NDINDDEKEDSENDNLKKYRQEIANLFNKSKYKSWVNVGKDGDKISGML 593FEKGLIDLLRTHFSDNLNEDIEIPELFSNKKIKDTRKLKEIINSFGKDGKDGQNFTTYFSVSFHNNRKNYYKSDGKMGRVSTRIVDENLERFCKNIYLYKEIIGKNEIKEIFSGNWDIYLQKKPNFSNDKTYKKLDEFKNDKYDWEMIFRDVNSYNKYFLQSDIEFYNYIRGKLNQDINEYNGKKRDSKEKINSQFENLRNQVHGEKKNYDDDFEIDEDNIIQFINEIFVRHNQNKMRFSEKLFSDFIDLLMVDNGDKLDKVYFSQKAVENAIARYYFVEETTNEGREPLLISLLLQNAGKDRKKLSNKPIKLGDIKFVLDQANNKPAEDIFKNRYVLSESNNDGIINANDKNHWANLLRLIKKDFYFHKDNLIKSQDKLALETKYNKGSDEGERQIETIKNFAESAKAILRMTKYFDLRKNGVIQNVIGGKDPIHEEVDKYFDGDVLSGEESCRISKYYDALRNFITKKAWSADKIILNFDCSEFLGGWDRSQEQKKRGIILRHRDGDEERYYLAVLGKNGKQYFENRTLFKGCESSDWQKIEYNVIQKPHMSLPKNLITPFFKKDKITNERFIDRSKKGAKALIEIDINPSDEFLNNYNLGKHTKENLDKSFLCDYFKYLMDAIAKYYKGEFNFNFPDVSNFDNTQPFYSFIEKNAYSIKYFGISSKEIEKLIADCYYKEDVYLFQIYCKDFEIDPKIGKAKYGNEFRTKAEIRKSKGEEAGNENLNTKYFKLLFDEKNLKNQNGIVYKLNGGAKMFYRPSSIKKDEKIDGKWRYKEDKYSLNITITCNFSSKKDDLSIDKDINKKIAEVNANSDFRIISIDRGEKNLAYCCVMDENANILDIKSLNRITRYDKNGKAIKEKNMFHEVKDGKLCYGEPVYDFYKDYQNLLDEREIKRLVNRRSWNVIEDIKNLKKGYVALLINYICKAVVIAINEGKYPIIVLESLDKGMLHNRVKIEKQIYRGVEEGLVRKLNYFVDKKTDNVLNAWQLLAKFETVGSSLDRKKQLGIIFYVDPGYTSITCPCCGFRQRKYIKAERAEENFKEIKIKFDGKRYSFAYDYRCIDDNGKEKSKEDIIYSNVKRLLRSGRNGRAVQIEDVTDELTNLFKKHNINIEQDINEQLAGKDNKFWKQLLWWFNAIEQIRNTQSLRRKFNTEENKLEILENNDCDFILCPHCYFDSNKDKFQNKIWNGDANGAFNIGRKGIIDIFEIKKHQRMLSDFMEQWGIDKLPKANGGNQAVIEIVKNDKKYNLCILNNKKIPYYCLRIGKEKIDSIADDRKCNQLPDLMVNWKKWDMWLDKWGK SEQ Cas12MPEVKNVFQDFTNLYELSKTLRFELKPVPETEKILELNAAKTKKFPKDLY ID variantRAENFEIIKKYTDELHRTYIRETLNNVNIDYLKFLEIFRINGKKKNEMTDEN NO:EESDENNEKDDIQKIKKELRSKIGNLFNKWNNDKDNKFKDWVKIDVGKK 594EKEVSGDLFGKELITILKNYFKNKLDSKVNVPMLFFNEQEIKNGEAKKQRKLEAVFENFDKFTTYFTDSFYNNRKNYYKTEGRVGQVATRIIDENLPRFCSNLIAFNEVVSLYSTLLNNFDLGWKEYLNEKKINQTWVEKFELSNYDWKALFNDVNYYNQCLLQEGIDKYNYIIKKLNKDINEYTQNKYKSVEKGNNNNPDINFFQKLHKQIHGERDFKLIEIDIDENNIFTKILPEFILHSDMKLMTKIDEEVGVEEIVGAERIIKIFIKQELKDLEKIYLSRRAIETISAKWFHSWETLKDLILGYLNKDLLESKKRKKVPDFVDFNIIKIVLENNKDDYKDLFKRKYFEADKNEFVDWIDSSGGTKKLEFGGENWINFLNVFEYEFGTLLTEYKKNKNALLYLIDKKIDYDKNNEVGQTAAIKNFADSALGIFRMVSYFALRKKGVMVEPKNGKDEIFYAFVDRYLDGDDNDREEQNKIVQYYNTLRNFVTQKAWSIDKVRLCFDCGEFLKGWDKDKIHERLGIILRNNNKFYLGILNKNHKQIFIKIKSHDNNNFYYVIYDYKQLNNVYRQIPRLAFPSRSVKKGDAYMLRAIQERKKKFFLEDEEFIELQEIKNEYDKIGNDLSKEKLTKLIEYYKKVVISNYSSLYNVSNLNNKKFNSINEFNQYVENLMYSLIPTRISPDFIKEKISKGELYLFQIYNKDFELDESIGKEKFGEDFAPVIMDGKNNLHTEYFKLLFNDSNLKNPNGVVFKLSGGAKMFYRPATENLPIKKDRDGNIIKNKKGENVIVGQRYKEDKYFLHLPIILNFVNKGKNYSINDMVNKAITNASDDQDKFRIIGLDRGEKHLVYYSVINERQEIIEIGSLNNISRKDNKGEIIEEKNWYHDKFGNIEKEPTKEYHKDYHNLLDQREIERLKSRQSWEKIENIKELKEGYISAVINKICNLVIKAIKENKIPIVALENLNSGMKRGRIKIDKQIYQKLELKLAKKLNFLVDKKEKNYLSAWQFTPKIETFSGDIEKKNQVGIIFYVDPAFTSATCPNCGFRKRIKMDPQNAKKKIKDMEITYENGIYKFDYPIENGENDVVYSDVERLKWDNEKKKVIKTKNVSDDFGKLFEDIKDKNNLKKELLSIGEENKEFWKEFSRCFNLLLRIRNSKLIKRKLNDDTGKVEIIADDDLADRDRDFIYCPQCHFHSEGGDVFGEFVKKKYLGKDNFEFNGDANGAYNIARKTIIAVNKIKDYQLGLNHFIEKYRISELPNNGKDKKNIFYNNNSYILSFFEVQDEKFRKVKVYGLKKDGDRQIIQKKEMWY RRYPDIFVNNKEWDKFVQNKSSEQ Cas12 MLFFMSTDITNKPREKGVFDNFTNLYEFSKTLTFGLIPLKWDDNKKMIVE ID variantDEDFSVLRKYGVIEEDKRIAESIKIAKFYLNILHRELIGKVLGSLKFEKKNL NO:ENYDRLLGEIEKNNKNENISEDKKKEIRKNFKKELSIAQDILLKKVGEVFE 595SNGSGILSSKNCLDELTKRFTRQEVDKLRRENKDIGVEYPDVAYREKDGKEETKSFFAMDVGYLDDFHKNRKQLYSVKGKKNSLGRRILDNFEIFCKNKKLYEKYKNLDIDFSEIERNFNLTLEKVFDFDNYNERLTQEGLDEYAKILGGESNKQERTANIHGLNQIINLYIQKKQSEQKAEQKETGKKKIKFNKKDYPTFTCLQKQILSQVFRKEIIIESDRDLIRELKFFVEESKEKVDKARGIIEFLLNHEENDIDLAMVYLPKSKINSFVYKVFKEPQDFLSVFQDGASNLDFVSFDKIKTHLENNKLTYKIFFKTLIKENHDFESFLILLQQEIDLLIDGGETVTLGGKKESITSLDEKKNRLKEKLGWFEGKVRENEKMKDEEEGEFCSTVLAYSQAVLNITKRAEIFWLNEKQDAKVGEDNKDMIFYKKFDEFADDGFAPFFYFDKFGNYLKRRSRNTTKEIKLHFGNDDLLEGWDMNKEPEYWSFILRDRNQYYLGIGKKDGEIFHKKLGNSVEAVKEAYELENEADFYEKIDYKQLNIDRFEGIAFPKKTKTEEAFRQVCKKRADEFLGGDTYEFKILLAIKKEYDDFKARRQKEKDWDSKFSKEKMSKLIEYYITCLGKRDDWKRFNLNFRQPKEYEDRSDFVRHIQRQAYWIDPRKVSKDYVDKKVAEGEMFLFKVHNKDFYDFERKSEDKKNHTANLFTQYLLELFSCENIKNIKSKDLIESIFELDGKAEIRFRPKTDDVKLKIYQKKGKDVTYADKRDGNKEKEVIQHRRFAKDALTLHLKIRLNFGKHVNLFDFNKLVNTELFAKVPVKILGMDRGENNLIYYCFLDEHGEIENGKCGSLNRVGEQIITLEDDKKVKEPVDYFQLLVDREGQRDWEQKNWQKMTRIKDLKKAYLGNVVSWISKEMLSGIKEGVVTIGVLEDLNSNFKRTRFFRERQVYQGFEKALVNKLGYLVDKKYDNYRNVYQFAPIVDSVEEMEKNKQIGTLVYVPASYTSKICPHPKCGWRERLYMKNSASKEKIVGLLKSDGIKISYDQKNDRFYFEYQWEQEHKSDGKKKKYSGVDKVFSNVSRMRWDVEQKKSIDFVDGTDGSITNKLKSLLKGKGIELDNINQQIVNQQKELGVEFFQSIIFYFNLIMQIRNYDKEKSGSEADYIQCPSCLFDSRKPEMNGKLSAITNGDANGAYNIARKGFMQLCRIRENPQEPMKLITNREWDEAVREWDIYSAAQKIPVLSEEN SEQ Cas12MTIKKHKPFTNFECLTPVQKTLRFRLIPVGRTTEFVKCRNIIEADRKRSEM ID variantYPLLKELADRFYREFMTDQLSNLLFDWSPLVEALLLARNNTDPRENQRIA NO:SLVRDEQKKYRTLLLKRLSGQVDRNGTPLPKNTASVNKKYYDDLFKARF 596VTETLPAYLEHLKNKPDGRISDELFDAYKDALDSYQKFTSRLTNFWQARKNIFTDEDIATGFAYRIVHEIVPDYLFNRRVYEQHKLDFPEPLDLLETELKKKNLIANDESLDALFTIPAINRLLTQKGVDLHNAVIGGFFTDDHTKVQGFNELANLKNQTLKNVSDNSEIKPVGKMTRLKKHILSISESTSFLFEQIESDDDLLARIIEFNNTLSEPDIDGLSIADINDQLYNIMTGVDPSTILVHARNLNKLSHEASLSWNRLRDGLYQMATESPYREDERFKRYIDASEEERDLSKLKNDIYFSLQELQFALDQSIDLEEEATPTEDIFLPFEFPGMDLKSELTVLFRSIEQLISSETKLIGNPDAIATIKKYLDAIMARYSIWNLLSCEAVELQDDLFYPEYDRVMGSLSNIILLYNLARNYLSRKPSSKEKFRLNFDKPTLADGWSESKVPDNFSVLLRKDDLFYLGILKDRKAYRVLSYENCDETAKNIKGYYERMIYHFSPDAYRMIPKCSTARKDVKKHFGEQGETTGYTLYPGASNFVKPFTIPYEIYRLQTELVNDKKRYQADYLKQTEDEEGYRQAVTAWIDFCKSYLESYEGTSTFDYSHLLKSEDYEDVNQFYADVDRASYSIYFEKVSVDLIHTMVDRGDLYLFQLYNKDFSPHSTGKPNLHTMYWRALFSNDNLQNNTIKLNGQAELFYRPKQVEQPTVHLQGSYLLNRFDKHGDVIPAGLYCEIYNHINERHPEGYTLSEEATQGLLDGRFVYREAPFELVKDKRYTEDQLFLHVPLEFNWTASANVPFENLANEYIKKDSDLHIIGIDRGERNLLYYSVINLQGDIVKQGSLNTLIQQTTLKGETVERQIPYQSMLKQREDERAEARQNWQSIDRIKDLKEGYLSHVIYKLSRLIIKYHAIVVMENLNVGFKRGRFKVERQVYQKFEVALINKLNALSFKEYEPNELGGVMRPWQLARRVVSPEDTRSQNGIVFYVPASYTSIVDPVTGFANLFYLNRIRNKDLNSFYGHFQEIRYDHEFDRFIFRFNYADFGVFCRIKNVPSRTWNLVSGERKAFNPKRRMIEKRDTTDEIKKALEAHGIAYQNEQNLLPLLLENENLLARIHRSFRLVLQLRNSDSDRDDIVSPALDKENNTFDSGQQPYESSLPINADANGAYNIARKGLLLVDKVKNDKRAVLSNREWFEYLMAEE SEQ Cas12MENKDYSLSRFTKQYQNSKTVRFALTPIGRTEEYIIQNQYIEAARRKNQA ID variantYKIVKPIIDEKFRSMIDDVLTHCEKQDWVTLDKLILQYQNNKCRENMDAL NO:AEQQEEIRKNISEEFTKSDEYKNFFGKEDSKKLFKIFLPEYLNQINASESDK 597EAVNEFQKFKTYFSNFLIVRADIFKADNKHNTIPYRIVNENFMIFAGNKRTFSNIIRLIPNALEEIAKDGMKKEEWSFYNIQNVDSWFEPDSFQMCMSQKGIQKYNFIIGLVNSYINLYTQQNPQATEVKRSRLKLRMLHKQILSDRVNPSWLPEQFKEGEEGEKQIYEAILALENDLIKNCFDKKYDLWIQSIDIQNPRIYIAASEMARVSSALHMGWNGLNDVRKTILLKSDKKQAKVEKILKQDVSLKDLSDTLNRYADIYKEEQIPSLYQYIEYGSELLQDCAITRKEYHDLLNGNSNTLSLNQNEKLIEGLKAYLDSYQAIVHFLNVFIVGDELDKDTDFYAELDGLVESLSEIVPLYNKVRNYITRKVYSLDKMRIMFERSDFLGGWGQSFDTKEALLFQKDNLYYIGIIEKKYTNMDVEYLHEGIKEGNRAIRFIYNFQKADNKNIPRTFIRSKGTNYAPAVRKYNLPIESIIDIYDVGKFKTNYKKINEKEYYESLEKLIDYFKDGILKNENYKKFHFNWKPSNEYENINEFYNDTNNACFLLEKEEINYDHLKEQANQGKIYLFQISSKDFNEGSKGTPNLQTMYWRELFSNQNCKDGVIKLCGGASIYMRDASIKQPVVHRKNAWLINKWYKVNGQNVVIPDNTYVKFTKIAQERMNEDELTPQERQLWNSGLIQKKKATHDIMKDRRFTKKQYMLHAPLTINYKQQDSPRYFNEKVRSFLKDNPDINIIGIDRGEKNLIYITIIDQKGNILKGMQKSFNQIEEKGKEGRTIDYYSKLESVEARHDAARKNWKQIGTIRELKEGYLSQVVHEITQLMIQYNAVIVMENLNMGFKKGRMKVEKSVYQKFEKMLIDKMNYLAFKRDMQGNAIDPYEVGGVMNGYQLTDRFTSFADMGSQNGFIFYVPAAYTSVIDPVTGFVNVFQKTEFKTNDFLHRFDSISWNDKEQSFVFTFDYQNFKCNGTCYQNKWSLYADVDRIETIIKNNQVDRIEPCNPNQKLIDFFDKKGIIYRDGHNIVDDLEKYDSKTISEIIHNFKLILQLRNSMRNPDTGEIIDYIASPVMHNEERFDSRKRNPELPQDADANGAYHIALKGLMFLQKINEYADSDGNMDNRKLKITNEEWFKYMQTRKEHTYF SEQ Cas12MSNKTSSITTTNKLSYTGFHNNGKQSKTLMFELKPIGRTTEHLDRKGYLA ID variantDDIDRAESYKTFKEIADNFHKNLIEESLATFTFSDTLKDYFDLWLSPVRTN NO:EDTPKLRKMEAKLRKELSSALKQHPSFAATSSGKRLIDEALYPNASDKER 598QCLDRFKGRSSYLDSYTEVRSFIYTDLCKHNTIAYRVVNENLKIYLENILAYEKLMQTAVNGKLETVKEMFHDLYPTFSMDISIFFTSYGFDYCLSQNAITRYNILLGGWSDDNGIHHKGLNNYINEYNQTVPRNKRLPKLNKLQKMILSEENSMSFIIDKFENDVDLANAIRYWLKNCQFDALNLLIWTLDVHYNLDEIHFKNDNQGKNISDLSQALFKNHHVIRDAWDYDYDIVNAKAKSRQKPERYAEKRDKAFKKINSFSLSYLANILSQYDNQYANFVAQFKTRISVHIQNVQQMIADKTLDMRLDPLMLLKSISSDTKLVEDIKRVLDSLKDMQRMLTPLLGEGTEPNRDAMFYSDFEPLMNYVDTLTPLYNKVRNYITKKPYSTKKTSLYFGASNFGSGFDVTKLPVSHTIIMRDKGCYYLAVIDNNKLIDKLYDHNDNDGYEYMVYKQIPSPIKYFSLKNILPQDPPDDIRQLLEDRKNGAKWSHDDETRFIDYIVNEFLPTYPPIHDKNGNPYFSWKFKNPDEYESLNEFFDDVSKQAYQTSFRFVSRDFVDDAVENGDIFFFQIYNQDFSPASHGKPSPHTLWFRALFSDVNLETKDIRLKGNATAYFRPASIFYTDEKWRKGHEIYEQLKNKFKFPIIKDKRYALDKFFFHITLEINCNATVEKYFNNRVNEEIRKADRYNILAINRGERNLLYAVVMDQDGTILEQKSFNIIKSELPNKTVKETDYWKKLHAREKERDTARKSWKSIECIKDLKKGYLSYVVKTITDMMFEYNAVLVMENLDIEMKRSRQKIEKNVYAQFQNAIIQKLSMYVNKDIDLHIARTAPGGTLNPYQLTYIPASRTKTPKQNGFVFFLNPWNITEIDPTTGFVDLFQTCFRTKNEYKDFFAKFKDIRYNEAQGWFEFDTDYTYFRDKEKAGKRTRWNICSYGTRLRRFRNPDKNYAEDAMTVYPTQMLKDLFDEYNIPYAPASAKSTSISIKDDIIQIDKLDFYKKLLYILKLIVQLRNTSPSSTEQEDDYIISPVINEDTNWFYDSRDYNEESLLPCNTDANGAYSLALKCNMVIDRIKNTIPGEPVDMYISNADWLDARQ SEQ Cas12MNSKTSIFDFSNIFGRDITLRFKLTPVTINSKGEVKDANGADPYRPYLSADE ID variantELQEQYELLKTAIDAYHQMYIDKKLKHILCLPLTEKGKDGVEHDTAKSKF NO:VKSCLAYIKDYGEKDKKRQTADLRTFISRVFADDNISSLPPYKVKSDFITK 599TLRQWLEQPDTKVEKKEAILDLIEKNGSKLYANCQGLLEARQRLYEKDGKSTSVPYRCIDRNLPRFSKDYHLFEKILGDCSDVFDFEQLDKDFSEELKGIARLSGIRVESVREVFQPLLYLAYLNQEGIQYLNTIIGTKKEKGTSALGLNEYINQYNQKQGIKKKKDGIPMLNKLNNQILFGDEVFIETLAEHKEAIPVIKKVVSSLGKLGAFDGECHENKLYQFLLSLSSYAGNIYVNTKVVAQISSSLWGDYSILYDAVKHDKNGRLIQKSVTLGELNEKIERLKLEDNRDAFEYFRRSQVKDVVHGSSNVGVFEQLKNCYNDFVEKKILKCSFFSEDQVLVIQRLFDSILSLQRIFKVFCPSLYEVDSDGLFVAKFSDYWNVLRGFDKDYDLLRNLFKRKPYSTDKIRVHFGLSNLMDGFVDSWTDKKDKGTQYNGYILRQAHSFVDENTSKELQEFQRYNYYLVISGNVRLFREKGNALVCEKKKEKLVASDEFSGFERFDYYQSSINNFNREFKRLTGRDRKSFTDEILQNEGKKELKSTYIENLIKVAKSMKRLTALQNLVSDEKVRKYSENLDYETLSAEIGQILATGRERKYVPVSTNEMKNLLKSSKNNKGEEVRTFMFRISNKDLSYAETMQKGERKSHGAENMHTMYFRALLDTLQNTFDIGTGTVYFRKASDKRKMKYDEKNPTHRKGDELAFKNPYNKGKKKSVFGYDLIKDRRYTKDSYLFHLSITQNYQKKGNAEDLNAMVRDYIRTQEDLRVIGIDRGERNLLYATMIDGEGHILAQKSFNVIGYQGTTASGESFQVETDYHQLLNEKAEKMRSLQREWKEMDKIQDMKDGYLSVVVHELAKMVVENNAIIVMEDLNMGFMESRQSQLANVYQKFEEKLRNKLQFYVDKRKRNDEPSGLYHALQLAGTETKDNQNGFIFYIPAWNTSKIDSVTGFVNLFNLKYTNIKDAKAFFSTFEKIEKNVETGHYDFTFSYSSMARKKMAKRMDGTRDSWTISTHGSRIVREQKGNYWEYREIESLTSEFDALFEKYSIDTRCRLKEAIDKCGEAEFFKELIRLMKWTLQLRNYDDRGNDYIVSPVCYRGNEYYCSLDYDNEEGMCISKIPCQMPKDADANGAFNIARKGLMLCERLKKGEKIGVIKGTEWLQYVQNMSERYVGMV SEQ Cas12MINTMEQPKKSIWDEFTNLYSLQKTLRFELKPQGKTKELVRTLFINPEEHH ID variantHKLISDDLELSKNYKKVKKLIDCMHRNIINNVLSKHQFTGEELKKLDKNS NO:NAEDNDTETDNADKKDPFAKIRERLTKALNEESKIMFDNKLLNPKKGKN 600KGECELKKWMDKAEDKYFELGNNEKIDKEAVKADMERLEGFFTYFGGFNKNRENVYSSKKIATAIPFRIIHDNFPIFKKNIENYKKITEKHPELAKLLNEKGANEIFQLEHFNKCLTQDGIDVYNNEKLGIIAKEQGKEQDKGINQLINEYAQKKNKEIKENAKGGEKPKKIKIAVFDKLKKQILSISKTKSFQFEVFEDTSDIINGINKRYTFLTEAKEGMSIVDEIKKIIGSVGDEKYSLDEIYLKEKFISTLSKKLFNYSRYIEVALEKWYDDRYDDKINKSGTDKRKFISAKQFSITSIQDAINYYLEKYEKDEELSKKYTGKNIIVDYFKNPTITIEHKQKEEVISEEKDLFKELEVRRNVIQHILNGDYKKDLKEEKQQDGDSEKVKAFLDALLEFNYILNPFIIKDKNLRKEQEKDEEFYNEIKKLQESIFEAEILDLYNQTRNYITKKPYKLDKFKLTFGSGYFLSGWSNDMEEREGSILIKYNEDRSKNYYLIIMAKPLTDDDKKQLFSDNGTHSKICIYEFQKMDMKNFPRMFINSKGSNPAPAIEKYNLPIKTIWADYQKYKNLNQKGKDKFLEENPDFRHNLIGYFKICAEKHESLAPFKHQFSSIWKPTKEYENLAQFYKDTLEACYNLKFENVNFDNISQLVSSGKLHLFKIHNKDFNPGSTGKKNLHTLYWEMLFDEKNLQDVIFKLSGGAELFYREASILKNKIIHKIGEKVLKKFFKLPDGKLEPVPAESIKNLSAYFRKELPEHELTEIDRKYIDNYSIIGKKDDKLGIMKDERFTVDKIQFHCPITINFKSKNKNFINDDVLEYLHKRDDVHIIGLDRGERHLIYLTMINKDGKIVDNMQFSLNELQRRYKINGNEEIQKINYQKLLDTREVSRTEARRNWQTIENIKNLKEGYLSLIVHQLAKLMIEKNAIVVMENLNYGFKDSRARVEKQIYQKFESILIKKLQYLVMDKNNLYDSGGVLSAYQLTNQEVPAYKYISKQNGFLFYVPPDYTSKIDPETGFINLLDTRYYSRKNAVALLNKFDKIYYDRDNKYFRFDFDYNSTDSNGNKNFDKLRVDISELTRTKWSVCSHPAKRSITVQINNKWVRQPINDVTDKLIKLFEDKQIGYESGKCLKDEILKVEDAKFFEDLLRYLSVLLALRHTYTENGVEYDLIISSVEKAPGSNEFFVSGKDNNLPANADANGAYNIARKGLWLLRKLDEIDNQELAIKKFNELKHAKEIKKNGEESKEDKGDRKRKKKWVSQWCPNK EWLAFAQSMQDVSEK SEQCas12 MNNGTNNFQNFIGISSLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQ ID variantILKDIMDDYYRGFISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQTE NO:YRKAIHKKFANDDRFKNMFSAKLISDILPEFVIHNNNYSASEKEEKTQVIK 601LFSRFATSFKDYFKNRANCFSADDISSSSCHRIVNDNAEIFFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKYGEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKFESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWETINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETYIHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEELVDKDNNFYAELEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYSNNAIILMRDNLYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSKTGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDTSTYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLHTMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQFGNIQIVRKNIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDKYFLHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKSFNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIAMEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKLKNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSEKNLFCFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTDINWRDGHDLRQDIIDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLISPVLNENNIFYDSAKAGDALPKDADANGAYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNKRYL SEQ Cas12MSNLNTFISPEFTGKIKMTKSLKVSMIPIGETEHWIAKHKVFEKDRELFDK ID variantNLKARPILDEFIKYTVSRALPNLLFDFEAYYLVKKDRTKARAFEKELAKT NO:VTDLILKEMDELKSASLIDSADFVKTTLKKFAGTHDIPGLSRIEAIESLEAA 602SKLTALNGKFNTSRIAIINTLIPKRIIENFDIYLSNMEKIRNVYESGEFGFLFERYPDTLLFMEPANYRTVCSPEAIEDYNRFISGYGDSTESWIKGFNQELSEASNSSKSSNGGVRRYSLIKPLHKQHLFETKKFFTFASISSDDDVRELINSVKGSTEDACLNALAFFSSSDPKTLFVKGSYLHTLSAFLYGSANSYILPERIKEGEKARLTAEYDSVAKKTKAVTTRYNVAMNNISKKINEKIFSLADIDAYCCDISKRRSVREILLGIMQEMYAAVYGENGKWSNIEAEAVLDSKTKIWKAKNGAVAKAVNDYLTAILEIRKFIRPFALRMEELEELGLDTSSALDAGEITNTLFEAVRAQKLVHAYLTRNDADIALSTQVYFGGTQKAAASWWNYETGDIQNRQIALAKKDGMYYFIGTFDERGSYSIEPASPGEDYYEMLDVKKGQDANKQIKKVLFSNKAIREHFADSSNDYVITTKVNSPITVRREIFDKYQAGEFKLTSQKIRKGDLVGEKEMTYYREYMDLLFQMAKGYTEYSRFNMDTLLPIEEYDTENDLLDDVNTNTIDYRWVRISAACIDDGVRNGDIFVFRAQTSSMYGKRENKKGYTGLFLELVSDENLLVTRGMSLNSAMSIYYRAKVHDAITVHKKGDVLVNKFTNARERIPENSYKAICAFYNSGKSIEELTIEDRDWLAKATTRICSGEIIKDRRYTKNQYSISISYNINRSVNNRKRVDLATIVDDTASAGRIISVTRGTKDLVYYTVIDDGGSVIEARSLNVINGINYAKMLAQISEERHDSNANFDIPKRVETIKEAYCAFAVHEIISAALKHNALIVVELISDAIKDKYSLLDNQVFLKFENVLKNCLMSVKVKGARGMEPGSISNPLQLCNADDKSFRNGILYQIPSSYINICPVTGYADIIDYYNIVSAGDIRNFFVRFENIVYNKEKARFEFSFDLKNIPIKLEKCPDRTKWTVLGRGEITTYDPLTKSNHYVFDAAQMLAETVSKEGLDPCANIVEHIDELSAATLKKMFNTFRNIAKGIVSECDEVPVSYYKSPVIDEADIKNKSLDNKSISEIKCYNLDEKARYYLALAKSSSDGENKNRYVSSTAIEW LNYIQEKRTHE

Alternatively, the Type V CRISPR/Cas enzyme is a programmable Cas14nuclease. A Cas14 protein of the present disclosure includes 3 partialRuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein assubdomains) that are not contiguous with respect to the primary aminoacid sequence of the Cas14 protein, but form a RuvC domain once theprotein is produced and folds. A naturally occurring Cas14 proteinfunctions as an endonuclease that catalyzes cleavage at a specificsequence in a target nucleic acid. A programmable Cas14 nuclease can bea Cas14a protein, a Cas114b protein, a Cas114c protein, a Cas114dprotein, a Cas114e protein, a Cas114f protein, a Cas114g protein, aCas114h protein, or a Cas114u protein. In some cases, a suitable Cas114protein comprises an amino acid sequence having at least 5000, at least5500, at least 60%, at least 65%, at least 70%, at least 7500 at least80%, at least 85%, at least 90%, at least 92%, at least 9500 at least9700, at least 9800, at least 990%, or 10000, amino acid sequenceidentity to any one of SEQ ID NO: 12-SEQ ID NO: 102.

TABLE 2 Cas14 Sequences SEQ ID NO Sequence SEQMEVQKTVMKTLSLRILRPLYSQEIEKEIKEEKERRKQAGGTGELDGGFYKKLEKKHSEM IDFSFDRLNLLLNQLQREIAKVYNHAISELYIATIAQGNKSNKHYISSIVYNRAYGYFYNAY NO:IALGICSKVEANFRSNELLTQQSALPTAKSDNFPIVLHKQKGAEGEDGGFRISTEGSDLIF 12EIPIPFYEYNGENRKEPYKWVKKGGQKPVLKLILSTFRRQRNKGWAKDEGTDAEIRKVTEGKYQVSQIEINRGKKLGEHQKWFANFSIEQPIYERKPNRSIVGGLDVGIRSPLVCAINNSFSRYSVDSNDVFKFSKQVFAFRRRLLSKNSLKRKGHGAAHKLEPITEMTEKNDKFRKKIIERWAKEVTNFFVKNQVGIVQIEDLSTMKDREDHFFNQYLRGFWPYYQMQTLIENKLKEYGIEVKRVQAKYTSQLCSNPNCRYWNNYFNFEYRKVNKFPKFKCEKCNLEISADYNAARNLSTPDIEKFVAKATKGINLPEK SEQMEEAKTVSKTLSLRILRPLYSAEIEKEIKEEKERRKQGGKSGELDSGFYKKLEKKHTQM IDFGWDKLNLMLSQLQRQIARVFNQSISELYIETVIQGKKSNKHYTSKIVYNRAYSVFYNA NO:YLALGITSKVEANFRSTELLMQKSSLPTAKSDNFPILLHKQKGVEGEEGGFKISADGNDL 13IFEIPIPFYEYDSANKKEPFKWIKKGGQKPTIKLILSTFRRQRNKGWAKDEGTDAEIRKVIEGKYQVSHIEINRGKKLGDHQKWFVNFTIEQPIYERKLDKNIIGGIDVGIKSPLVCAVNNSFARYSVDSNDVLKFSKQAFAFRRRLLSKNSLKRSGHGSKNKLDPITRMTEKNDRFRKKIIERWAKEVTNFFIKNQVGTVQIEDLSTMKDRQDNFFNQYLRGFWPYYQMQNLIENKLKEYGIETKRIKARYTSQLCSNPSCRHWNSYFSFDHRKTNNFPKFKCEKCALEISADYNAARNISTPDIEKFVAKATKGINLPDKNENVILE SEQMAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAA IDYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSL NO:IELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKN 14MKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIDVGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP SEQMERQKVPQIRKIVRVVPLRILRPKYSDVIENALKKFKEKGDDTNTNDFWRAIRDRDTEF IDFRKELNFSEDEINQLERDTLFRVGLDNRVLFSYFDFLQEKLMKDYNKIISKLFINRQSKSS NO:FENDLTDEEVEELIEKDVTPFYGAYIGKGIKSVIKSNLGGKFIKSVKIDRETKKVTKLTAI 15NIGLMGLPVAKSDTFPIKIIKTNPDYITFQKSTKENLQKIEDYETGIEYGDLLVQITIPWFKNENKDFSLIKTKEAIEYYKLNGVGKKDLLNINLVLTTYHIRKKKSWQIDGSSQSLVREMANGELEEKWKSFFDTFIKKYGDEGKSALVKRRVNKKSRAKGEKGRELNLDERIKRLYDSIKAKSFPSEINLIPENYKWKLHFSIEIPPMVNDIDSNLYGGIDFGEQNIATLCVKNIEKDDYDFLTIYGNDLLKHAQASYARRRIMRVQDEYKARGHGKSRKTKAQEDYSERMQKLRQKITERLVKQISDFFLWRNKFHMAVCSLRYEDLNTLYKGESVKAKRMRQFINKQQLFNGIERKLKDYNSEIYVNSRYPHYTSRLCSKCGKLNLYFDFLKFRTKNIIIRKNPDGSEIKYMPFFICEFCGWKQAGDKNASANIADKDYQDKLNKEKEFCNIRKPKSKKEDIGEENEEERDYSRRFNRNSFIYNSLKKDNKLNQEKLFDEWKNQLKRKIDGRNKFEPKEYKDRFSYLFAYY QEIIKNESESSEQ MVPTELITKTLQLRVIRPLYFEEIEKELAELKEQKEKEFEETNSLLLESKKIDAKSLKKLK IDRKARSSAAVEFWKIAKEKYPDILTKPEMEFIFSEMQKMMARFYNKSMTNIFIEMNNDEK NO:VNPLSLISKASTEANQVIKCSSISSGLNRKIAGSINKTKFKQVRDGLISLPTARTETFPISFY 16KSTANKDEIPISKINLPSEEEADLTITLPFPFFEIKKEKKGQKAYSYFNIIEKSGRSNNKIDLLLSTHRRQRRKGWKEEGGTSAEIRRLMEGEFDKEWEIYLGEAEKSEKAKNDLIKNMTRGKLSKDIKEQLEDIQVKYFSDNNVESWNDLSKEQKQELSKLRKKKVEELKDWKHVKEILKTRAKIGWVELKRGKRQRDRNKWFVNITITRPPFINKELDDTKFGGIDLGVKVPFVCAVHGSPARLIIKENEILQFNKMVSARNRQITKDSEQRKGRGKKNKFIKKEIFNERNELFRKKIIERWANQIVKFFEDQKCATVQIENLESFDRTSYK SEQMKSDTKDKKIIIHQTKTLSLRIVKPQSIPMEEFTDLVRYHQMIIFPVYNNGAIDLYKKLFK IDAKIQKGNEARAIKYFMNKIVYAPIANTVKNSYIALGYSTKMQSSFSGKRLWDLRFGEAT NO:PPTIKADFPLPFYNQSGFKVSSENGEFIIGIPFGQYTKKTVSDIEKKTSFAWDKFTLEDTTK 17KTLIELLLSTKTRKMNEGWKNNEGTEAEIKRVMDGTYQVTSLEILQRDDSWFVNFNIAYDSLKKQPDRDKIAGIHMGITRPLTAVIYNNKYRALSIYPNTVMHLTQKQLARIKEQRTNSKYATGGHGRNAKVTGTDTLSEAYRQRRKKIIEDWIASIVKFAINNEIGTIYLEDISNTNSFFAAREQKLIYLEDISNTNSFLSTYKYPISAISDTLQHKLEEKAIQVIRKKAYYVNQICSLCGHYNKGFTYQFRRKNKFPKMKCQGCLEATSTEFNAAANVANPDYEKLLIKHGLLQL KK SEQMSTITRQVRLSPTPEQSRLLMAHCQQYISTVNVLVAAFDSEVLTGKVSTKDFRAALPSA IDVKNQALRDAQSVFKRSVELGCLPVLKKPHCQWNNQNWRVEGDQLILPICKDGKTQQE NO:RFRCAAVALEGKAGILRIKKKRGKWIADLTVTQEDAPESSGSAIMGVDLGIKVPAVAHI 18GGKGTRFFGNGRSQRSMRRRFYARRKTLQKAKKLRAVRKSKGKEARWMKTINHQLSRQIVNHAHALGVGTIKIEALQGIRKGTTRKSRGAAARKNNRMTNTWSFSQLTLFITYKAQRQGITVEQVDPAYTSQDCPACRARNGAQDRTYVCSECGWRGHRDTVGAINISRRAGLS GHRRGATGA SEQMIAQKTIKIKLNPTKEQIIKLNSIIEEYIKVSNFTAKKIAEIQESFTDSGLTQGTCSECGKEK IDTYRKYHLLKKDNKLFCITCYKRKYSQFTLQKVEFQNKTGLRNVAKLPKTYYTNAIRFA NO:SDTFSGFDEIIKKKQNRLNSIQNRLNFWKELLYNPSNRNEIKIKVVKYAPKTDTREHPHY 19YSEAEIKGRIKRLEKQLKKFKMPKYPEFTSETISLQRELYSWKNPDELKISSITDKNESMNYYGKEYLKRYIDLINSQTPQILLEKENNSFYLCFPITKNIEMPKIDDTFEPVGIDWGITRNIAVVSILDSKTKKPKFVKFYSAGYILGKRKHYKSLRKHFGQKKRQDKINKLGTKEDRFIDSNIHKLAFLIVKEIRNHSNKPIILMENITDNREEAEKSMRQNILLHSVKSRLQNYIAYKALWNNIPTNLVKPEHTSQICNRCGHQDRENRPKGSKLFKCVKCNYMSNADFNASINIARKFYIGEYEPFYKDNEKMKSGVNSISM SEQLKLSEQENITTGVKFKLKLDKETSEGLNDYFDEYGKAINFAIKVIQKELAEDRFAGKVRL IDDENKKPLLNEDGKKIWDFPNEFCSCGKQVNRYVNGKSLCQECYKNKFTEYGIRKRMYS NO:AKGRKAEQDINIKNSTNKISKTHFNYAIREAFILDKSIKKQRKERFRRLREMKKKLQEFIE 20IRDGNKILCPKIEKQRVERYIHPSWINKEKKLEDFRGYSMSNVLGKIKILDRNIKREEKSLKEKGQINFKARRLMLDKSVKFLNDNKISFTISKNLPKEYELDLPEKEKRLNWLKEKIKIIKNQKPKYAYLLRKDDNFYLQYTLETEFNLKEDYSGIVGIDRGVSHIAVYTFVHNNGKNERPLFLNSSEILRLKNLQKERDRFLRRKHNKKRKKSNMRNIEKKIQLILHNYSKQIVDFAKNKNAFIVFEKLEKPKKNRSKMSKKSQYKLSQFTFKKLSDLVDYKAKREGIKVLYISPEYTSKECSHCGEKVNTQRPFNGNSSLFKCNKCGVELNADYNASINIAKKGLNILNSTN SEQMEESIITGVKFKLRIDKETTKKLNEYFDEYGKAINFAVKIIQKELADDRFAGKAKLDQNK IDNPILDENGKKIYEFPDEFCSCGKQVNKYVNNKPFCQECYKIRFTENGIRKRMYSAKGRK NO:AEHKINILNSTNKISKTHFNYAIREAFILDKSIKKQRKKRNERLRESKKRLQQFIDMRDG 21KREICPTIKGQKVDRFIHPSWITKDKKLEDFRGYTLSIINSKIKILDRNIKREEKSLKEKGQIIFKAKRLMLDKSIRFVGDRKVLFTISKTLPKEYELDLPSKEKRLNWLKEKIEIIKNQKPKYAYLLRKNIESEKKPNYEYYLQYTLEIKPELKDFYDGAIGIDRGINHIAVCTFISNDGKVTPPKFFSSGEILRLKNLQKERDRFLLRKHNKNRKKGNMRVIENKINLILHRYSKQIVDMAKKLNASIVFEELGRIGKSRTKMKKSQRYKLSLFIFKKLSDLVDYKSRREGIRVTYVPPEYTSKECSHCGEKVNTQRPFNGNYSLFKCNKCGIQLNSDYNASINIAKKGLKIPNST SEQLWTIVIGDFIEMPKQDLVTTGIKFKLDVDKETRKKLDDYFDEYGKAINFAVKIIQKNLKE IDDRFAGKIALGEDKKPLLDKDGKKIYNYPNESCSCGNQVRRYVNAKPFCVDCYKLKFTE NO:NGIRKRMYSARGRKADSDINIKNSTNKISKTHFNYAIREGFILDKSLKKQRSKRIKKLLEL 22KRKLQEFIDIRQGQMVLCPKIKNQRVDKFIHPSWLKRDKKLEEFRGYSLSVVEGKIKIFNRNILREEDSLRQRGHVNFKANRIMLDKSVRFLDGGKVNFNLNKGLPKEYLLDLPKKENKLSWLNEKISLIKLQKPKYAYLLRREGSFFIQYTIENVPKTFSDYLGAIGIDRGISHIAVCTFVSKNGVNKAPVFFSSGEILKLKSLQKQRDLFLRGKHNKIRKKSNMRNIDNKINLILHKYSRNIVNLAKSEKAFIVFEKLEKIKKSRFKMSKSLQYKLSQFTFKKLSDLVEYKAKIEGIKVDYVPPEYTSKECSHCGEKVDTQRPFNGNSSLFKCNKCRVQLNADYNASINIAKKSLNI SN SEQMSKTTISVKLKIIDLSSEKKEFLDNYFNEYAKATTFCQLRIRRLLRNTHWLGKKEKSSKK IDWIFESGICDLCGENKELVNEDRNSGEPAKICKRCYNGRYGNQMIRKLFVSTKKREVQEN NO:MDIRRVAKLNNTHYHRIPEEAFDMIKAADTAEKRRKKNVEYDKKRQMEFIEMFNDEK 23KRAARPKKPNERETRYVHISKLESPSKGYTLNGIKRKIDGMGKKIERAEKGLSRKKIFGYQGNRIKLDSNWVRFDLAESEITIPSLFKEMKLRITGPTNVHSKSGQIYFAEWFERINKQPNNYCYLIRKTSSNGKYEYYLQYTYEAEVEANKEYAGCLGVDIGCSKLAAAVYYDSKNKKAQKPIEIFTNPIKKIKMRREKLIKLLSRVKVRHRRRKLMQLSKTEPIIDYTCHKTARKIVEMANTAKAFISMENLETGIKQKQQARETKKQKFYRNMFLFRKLSKLIEYKALLKGIKIVYVKPDYTSQTCSSCGADKEKTERPSQAIFRCLNPTCRYYQRDINADFNAAVNIAKKALN NTEVVTTLLSEQ MARAKNQPYQKLTTTTGIKFKLDLSEEEGKRFDEYFSEYAKAVNFCAKVIYQLRKNLK IDFAGKKELAAKEWKFEISNCDFCNKQKEIYYKNIANGQKVCKGCHRTNFSDNAIRKKMI NO:PVKGRKVESKFNIHNTTKKISGTHRHWAFEDAADIIESMDKQRKEKQKRLRREKRKLSY 24FFELFGDPAKRYELPKVGKQRVPRYLHKIIDKDSLTKKRGYSLSYIKNKIKISERNIERDEKSLRKASPIAFGARKIKMSKLDPKRAFDLENNVFKIPGKVIKGQYKFFGTNVANEHGKKFYKDRISKILAGKPKYFYLLRKKVAESDGNPIFEYYVQWSIDTETPAITSYDNILGIDAGITNLATTVLIPKNLSAEHCSHCGNNHVKPIFTKFFSGKELKAIKIKSRKQKYFLRGKHNKLVKIKRIRPIEQKVDGYCHVVSKQIVEMAKERNSCIALEKLEKPKKSKFRQRRREKYAVSMFVFKKLATFIKYKAAREGIEIIPVEPEGTSYTCSHCKNAQNNQRPYFKPNSKKSWTSMFKCGKCGIELNSDYNAAFNIAQKALNMTSA SEQMDEKHFFCSYCNKELKISKNLINKISKGSIREDEAVSKAISIHNKKEHSLILGIKFKLFIEN IDKLDKKKLNEYFDNYSKAVTFAARIFDKIRSPYKFIGLKDKNTKKWTFPKAKCVFCLEEK NO:EVAYANEKDNSKICTECYLKEFGENGIRKKIYSTRGRKVEPKYNIFNSTKELSSTHYNYA 25IRDAFQLLDALKKQRQKKLKSIFNQKLRLKEFEDIFSDPQKRIELSLKPHQREKRYIHLSKSGQESINRGYTLRFVRGKIKSLTRNIEREEKSLRKKTPIHFKGNRLMIFPAGIKFDFASNKVKISISKNLPNEFNFSGTNVKNEHGKSFFKSRIELIKTQKPKYAYVLRKIKREYSKLRNYEIEKIRLENPNADLCDFYLQYTIETESRNNEEINGIIGIDRGITNLACLVLLKKGDKKPSGVKFYKGNKILGMKIAYRKHLYLLKGKRNKLRKQRQIRAIEPKINLILHQISKDIVKIAKEKNFAIALEQLEKPKKARFAQRKKEKYKLALFTFKNLSTLIEYKSKREGIPVIYVPPEKTSQMCSHCAINGDEHVDTQRPYKKPNAQKPSYSLFKCNKCGIELNADYNAAFNIAQKGLKTLM LNHSH SEQMLQTLLVKLDPSKEQYKMLYETMERFNEACNQIAETVFAIHSANKIEVQKTVYYPIREK IDFGLSAQLTILAIRKVCEAYKRDKSIKPEFRLDGALVYDQRVLSWKGLDKVSLVTLQGRQ NO:IIPIKFGDYQKARMDRIRGQADLILVKGVFYLCVVVEVSEESPYDPKGVLGVDLGIKNLA 26VDSDGEVHSGEQTTNTRERLDSLKARLQSKGTKSAKRHLKKLSGRMAKFSKDVNHCISKKLVAKAKGTLMSIALEDLQGIRDRVTVRKAQRRNLHTWNFGLLRMFVDYKAKIAGVPLVFVDPRNTSRTCPSCGHVAKANRPTRDEFRCVSCGFAGAADHIAAMNIAFRAEVSQPIVTRFFVQSQAPSFRVG SEQMDEEPDSAEPNLAPISVKLKLVKLDGEKLAALNDYFNEYAKAVNFCELKMQKIRKNLV IDNIRGTYLKEKKAWINQTGECCICKKIDELRCEDKNPDINGKICKKCYNGRYGNQMIRKL NO:FVSTNKRAVPKSLDIRKVARLHNTHYHRIPPEAADIIKAIETAERKRRNRILFDERRYNEL 27KDALENEEKRVARPKKPKEREVRYVPISKKDTPSKGYTMNALVRKVSGMAKKIERAKRNLNKRKKIEYLGRRILLDKNWVRFDFDKSEISIPTMKEFFGEMRFEITGPSNVMSPNGREYFTKWFDRIKAQPDNYCYLLRKESEDETDFYLQYTWRPDAHPKKDYTGCLGIDIGGSKLASAVYFDADKNRAKQPIQIFSNPIGKWKTKRQKVIKVLSKAAVRHKTKKLESLRNIEPRIDVHCHRIARKIVGMALAANAFISMENLEGGIREKQKAKETKKQKFSRNMFVFRKLSKLIEYKALMEGVKVVYIVPDYTSQLCSSCGTNNTKRPKQAIFMCQNTECRYFGKNINADFNAAINIAKKALNRKDIVRELS SEQMEKNNSEQTSITTGIKFKLKLDKETKEKLNNYFDEYGKAINFAVRIIQMQLNDDRLAGK IDYKRDEKGKPILGEDGKKILEIPNDFCSCGNQVNHYVNGVSFCQECYKKRFSENGIRKRM NO:YSAKGRKAEQDINIKNSTNKISKTHFNYAIREAFNLDKSIKKQREKRFKKLKDMKRKLQ 28EFLEIRDGKRVICPKIEKQKVERYIHPSWINKEKKLEEFRGYSLSIVNSKIKSFDRNIQREEKSLKEKGQINFKAQRLMLDKSVKFLKDNKVSFTISKELPKTFELDLPKKEKKLNWLNEKLEIIKNQKPKYAYLLRKENNIFLQYTLDSIPEIHSEYSGAVGIDRGVSHIAVYTFLDKDGKNERPFFLSSSGILRLKNLQKERDKFLRKKHNKIRKKGNMRNIEQKINLILHEYSKQIVNFAKDKNAFIVFELLEKPKKSRERMSKKIQYKLSQFTFKKLSDLVDYKAKREGIKVIYVEPAYTSKDCSHCGERVNTQRPFNGNFSLFKCNKCGIVLNSDYNASLNIARKGLNISAN SEQMAEEKFFFCEKCNKDIKIPKNYINKQGAEEKARAKHEHRVHALILGIKFKIYPKKEDISK IDLNDYFDEYAKAVTFTAKIVDKLKAPFLFAGKRDKDTSKKKWVFPVDKCSFCKEKTEIN NO:YRTKQGKNICNSCYLTEFGEQGLLEKIYATKGRKVSSSFNLFNSTKKLTGTHNNYVVKE 29SLQLLDALKKQRSKRLKKLSNTRRKLKQFEEMFEKEDKRFQLPLKEKQRELRFIHVSQKDRATEFKGYTMNKIKSKIKVLRRNIEREQRSLNRKSPVFFRGTRIRLSPSVQFDDKDNKIKLTLSKELPKEYSFSGLNVANEHGRKFFAEKLKLIKENKSKYAYLLRRQVNKNNKKPIYDYYLQYTVEFLPNIITNYNGILGIDRGINTLACIVLLENKKEKPSFVKFFSGKGILNLKNKRRKQLYFLKGVHNKYRKQQKIRPIEPRIDQILHDISKQIIDLAKEKRVAISLEQLEKPQKPKFRQSRKAKYKLSQFNFKTLSNYIDYKAKKEGIRVIYIAPEMTSQNCSRCAMKNDLHVNTQRPYKNTSSLFKCNKCGVELNADYNAAFNIAQKGLKILNS SEQMISLKLKLLPDEEQKKLLDEMFWKWASICTRVGFGRADKEDLKPPKDAEGVWFSLTQL IDNQANTDINDLREAMKHQKHRLEYEKNRLEAQRDDTQDALKNPDRREISTKRKDLFRPK NO:ASVEKGFLKLKYHQERYWVRRLKEINKLIERKTKTLIKIEKGRIKFKATRITLHQGSFKIR 30FGDKPAFLIKALSGKNQIDAPFVVVPEQPICGSVVNSKKYLDEITTNFLAYSVNAMLFGLSRSEEMLLKAKRPEKIKKKEEKLAKKQSAFENKKKELQKLLGRELTQQEEAIIEETRNQFFQDFEVKITKQYSELLSKIANELKQKNDFLKVNKYPILLRKPLKKAKSKKINNLSPSEWKYYLQFGVKPLLKQKSRRKSRNVLGIDRGLKHLLAVTVLEPDKKTFVWNKLYPNPITGWKWRRRKLLRSLKRLKRRIKSQKHETIHENQTRKKLKSLQGRIDDLLHNISRKIVETAKEYDAVIVVEDLQSMRQHGRSKGNRLKTLNYALSLFDYANVMQLIKYKAGIEGIQIYDVKPAGTSQNCAYCLLAQRDSHEYKRSQENSKIGVCLNPNCQNHKKQIDADLNAARVIASCYALKINDSQPFGTRKRFKKRTTN SEQMETLSLKLKLNPSKEQLLVLDKMFWKWASICTRLGLKKAEMSDLEPPKDAEGVWFSK IDTQLNQANTDVNDLRKAMQHQGKRIEYELDKVENRRNEIQEMLEKPDRRDISPNRKDLF NO:RPKAAVEKGYLKLKYHKLGYWSKELKTANKLIERKRKTLAKIDAGKMKFKPTRISLHT 31NSFRIKFGEEPKIALSTTSKHEKIELPLITSLQRPLKTSCAKKSKTYLDAAILNFLAYSTNAALFGLSRSEEMLLKAKKPEKIEKRDRKLATKRESFDKKLKTLEKLLERKLSEKEKSVFKRKQTEFFDKFCITLDETYVEALHRIAEELVSKNKYLEIKKYPVLLRKPESRLRSKKLKNLKPEDWTYYIQFGFQPLLDTPKPIKTKTVLGIDRGVRHLLAVSIFDPRTKTFTFNRLYSNPIVDWKWRRRKLLRSIKRLKRRLKSEKHVHLHENQFKAKLRSLEGRIEDHFHNLSKEIVDLAKENNSVIVVENLGGMRQHGRGRGKWLKALNYALSHFDYAKVMQLIKYKAELAGVFVYDVAPAGTSINCAYCLLNDKDASNYTRGKVINGKKNTKIGECKTCKKEFDADLNAARVIALCYEKRLNDPQPFGTRKQFKPKKP SEQMKALKLQLIPTRKQYKILDEMFWKWASLANRVSQKGESKETLAPKKDIQKIQFNATQL IDNQIEKDIKDLRGAMKEQQKQKERLLLQIQERRSTISEMLNDDNNKERDPHRPLNFRPKG NO:WRKFHTSKHWVGELSKILRQEDRVKKTIERIVAGKISFKPKRIGIWSSNYKINFFKRKISI 32NPLNSKGFELTLMTEPTQDLIGKNGGKSVLNNKRYLDDSIKSLLMFALHSRFFGLNNTDTYLLGGKINPSLVKYYKKNQDMGEFGREIVEKFERKLKQEINEQQKKIIMSQIKEQYSNRDSAFNKDYLGLINEFSEVFNQRKSERAEYLLDSFEDKIKQIKQEIGESLNISDWDFLIDEAKKAYGYEEGFTEYVYSKRYLEILNKIVKAVLITDIYFDLRKYPILLRKPLDKIKKISNLKPDEWSYYIQFGYDSINPVQLMSTDKFLGIDRGLTHLLAYSVFDKEKKEFIINQLEPNPIMGWKWKLRKVKRSLQHLERRIRAQKMVKLPENQMKKKLKSIEPKIEVHYHNISRKIVNLAKDYNASIVVESLEGGGLKQHGRKKNARNRSLNYALSLFDYGKIASLIKYKADLEGVPMYEVLPAYTSQQCAKCVLEKGSFVDPEIIGYVEDIGIKGSLLDSLFEGTELSSIQVLKKIKNKIELSARDNHNKEINLILKYNFKGLVIVRGQDKEEIAEHPIKEINGKFAILDFVYKRGKEKVGKKGNQKVRYTGNKKVGYCSKHGQVDADLNASRVIALCKYLDINDPILFGEQRKSF K SEQMVTRAIKLKLDPTKNQYKLLNEMFWKWASLANRFSQKGASKETLAPKDGTQKIQFNA IDTQLNQIKKDVDDLRGAMEKQGKQKERLLIQIQERLLTISEILRDDSKKEKDPHRPQNFRP NO:FGWRRFHTSAYWSSEASKLTRQVDRVRRTIERIKAGKINFKPKRIGLWSSTYKINFLKKK 33INISPLKSKSFELDLITEPQQKIIGKEGGKSVANSKKYLDDSIKSLLIFAIKSRLFGLNNKDKPLFENIITPNLVRYHKKGQEQENFKKEVIKKFENKLKKEISQKQKEIIFSQIERQYENRDATFSEDYLRAISEFSEIFNQRKKERAKELLNSFNEKIRQLKKEVNGNISEEDLKILEVEAEKAYNYENGFIEWEYSEQFLGVLEKIARAVLISDNYFDLKKYPILIRKPTNKSKKITNLKPEEWDYYIQFGYGLINSPMKIETKNFMGIDRGLTHLLAYSIFDRDSEKFTINQLELNPIKGWKWKLRKVKRSLQHLERRMRAQKGVKLPENQMKKRLKSIEPKIESYYHNLSRKIVNLAKANNASIVVESLEGGGLKQHGRKKNSRHRALNYALSLFDYGKIASLIKYKSDLEGVPMYEVLPAYTSQQCAKCVLKKGSFVEPEIIGYIEEIGFKENLLTLLFEDTGLSSVQVLKKSKNKMTLSARDKEGKMVDLVLKYNFKGLVISQEKKKEEIVEFPIKEIDGKFAVLDSAYKRGKERISKKGNQKLVYTGNKKVGYCSVHGQVDADLNASRVIALCKYLGINEPIVFGEQRKSF K SEQLDLITEPIQPHKSSSLRSKEFLEYQISDFLNFSLHSLFFGLASNEGPLVDFKIYDKIVIPKPE IDERFPKKESEEGKKLDSFDKRVEEYYSDKLEKKIERKLNTEEKNVIDREKTRIWGEVNKL NO:EEIRSIIDEINEIKKQKHISEKSKLLGEKWKKVNNIQETLLSQEYVSLISNLSDELTNKKKE 34LLAKKYSKFDDKIKKIKEDYGLEFDENTIKKEGEKAFLNPDKFSKYQFSSSYLKLIGEIARSLITYKGFLDLNKYPIIFRKPINKVKKIHNLEPDEWKYYIQFGYEQINNPKLETENILGIDRGLTHILAYSVFEPRSSKFILNKLEPNPIEGWKWKLRKLRRSIQNLERRWRAQDNVKLPENQMKKNLRSIEDKVENLYHNLSRKIVDLAKEKNACIVFEKLEGQGMKQHGRKKSDRLRGLNYKLSLFDYGKIAKLIKYKAEIEGIPIYRIDSAYTSQNCAKCVLESRRFAQPEEISCLDDFKEGDNLDKRILEGTGLVEAKIYKKLLKEKKEDFEIEEDIAMFDTKKVIKENKEKTVILDYVYTRRKEIIGTNHKKNIKGIAKYTGNTKIGYCMKHGQVDADLNASRTIALCKNFDI NNPEIWK SEQMSDESLVSSEDKLAIKIKIVPNAEQAKMLDEMFKKWSSICNRISRGKEDIETLRPDEGKE IDLQFNSTQLNSATMDVSDLKKAMARQGERLEAEVSKLRGRYETIDASLRDPSRRHTNPQ NO:KPSSFYPSDWDISGRLTPRFHTARHYSTELRKLKAKEDKMLKTINKIKNGKIVFKPKRIT 35LWPSSVNMAFKGSRLLLKPFANGFEMELPIVISPQKTADGKSQKASAEYMRNALLGLAGYSINQLLFGMNRSQKMLANAKKPEKVEKFLEQMKNKDANFDKKIKALEGKWLLDRKLKESEKSSIAVVRTKFFKSGKVELNEDYLKLLKHMANEILERDGFVNLNKYPILSRKPMKRYKQKNIDNLKPNMWKYYIQFGYEPIFERKASGKPKNIMGIDRGLTHLLAVAVFSPDQQKFLFNHLESNPIMHWKWKLRKIRRSIQHMERRIRAEKNKHIHEAQLKKRLGSIEEKTEQHYHIVSSKIINWAIEYEAAIVLESLSHMKQRGGKKSVRTRALNYALSLFDYEKVARLITYKARIRGIPVYDVLPGMTSKTCATCLLNGSQGAYVRGLETTKAAGKATKRKNMKIGKCMVCNSSENSMIDADLNAARVIAICKYKNLNDPQPAGSRKVFKRF SEQMLALKLKIMPTEKQAEILDAMFWKWASICSRIAKMKKKVSVKENKKELSKKIPSNSDI IDWFSKTQLCQAEVDVGDHKKALKNFEKRQESLLDELKYKVKAINEVINDESKREIDPNN NO:PSKFRIKDSTKKGNLNSPKFFTLKKWQKILQENEKRIKKKESTIEKLKRGNIFFNPTKISL 36HEEEYSINFGSSKLLLNCFYKYNKKSGINSDQLENKFNEFQNGLNIICSPLQPIRGSSKRSFEFIRNSIINFLMYSLYAKLFGIPRSVKALMKSNKDENKLKLEEKLKKKKSSFNKTVKEFEKMIGRKLSDNESKILNDESKKFFEIIKSNNKYIPSEEYLKLLKDISEEIYNSNIDFKPYKYSILIRKPLSKFKSKKLYNLKPTDYKYYLQLSYEPFSKQLIATKTILGIDRGLKHLLAVSVFDPSQNKFVYNKLIKNPVFKWKKRYHDLKRSIRNRERRIRALTGVHIHENQLIKKLKSMKNKINVLYHNVSKNIVDLAKKYESTIVLERLENLKQHGRSKGKRYKKLNYVLSNFDYKKIESLISYKAKKEGVPVSNINPKYTSKTCAKCLLEVNQLSELKNEYNRDSKNSKIGICNIHGQIDADLNAARVIALCYSKNLNEPHFK SEQVINLFGYKFALYPNKTQEELLNKHLGECGWLYNKAIEQNEYYKADSNIEEAQKKFELLP IDDKNSDEAKVLRGNISKDNYVYRTLVKKKKSEINVQIRKAVVLRPAETIRNLAKVKKKG NO:LSVGRLKFIPIREWDVLPFKQSDQIRLEENYLILEPYGRLKFKMHRPLLGKPKTFCIKRTA 37TDRWTISFSTEYDDSNMRKNDGGQVGIDVGLKTHLRLSNENPDEDPRYPNPKIWKRYDRRLTILQRRISKSKKLGKNRTRLRLRLSRLWEKIRNSRADLIQNETYEILSENKLIAIEDLNVKGMQEKKDKKGRKGRTRAQEKGLHRSISDAAFSEFRRVLEYKAKRFGSEVKPVSAIDSSKECHNCGNKKGMPLESRIYECPKCGLKIDRDLNSAKVILARATGVRPGSNARADTKISATAGASVQTEGTVSEDFRQQMETSDQKPMQGEGSKEPPMNPEHKSSGRGSKHVNIGCKNKVGLYNEDENSRSTEKQIMDENRSTTEDMVEIGALHSPVLTT SEQMIASIDYEAVSQALIVFEFKAKGKDSQYQAIDEAIRSYRFIRNSCLRYWMDNKKVGKYD IDLNKYCKVLAKQYPFANKLNSQARQSAAECSWSAISRFYDNCKRKVSGKKGFPKFKKH NO:ARSVEYKTSGWKLSENRKAITFTDKNGIGKLKLKGTYDLHFSQLEDMKRVRLVRRADG 38YYVQFCISVDVKVETEPTGKAIGLDVGIKYFLADSSGNTIENPQFYRKAEKKLNRANRRKSKKYIRGVKPQSKNYHKARCRYARKHLRVSRQRKEYCKRVAYCVIHSNDVVAYEDLNVKGMVKNRHLAKSISDVAWSTFRHWLEYFAIKYGKLTIPVAPHNTSQNCSNCDKKVPKSLSTRTHICHHCGYSEDRDVNAAKNILKKALSTVGQTGSLKLGEIEPLLVLEQSCTRKF DL SEQLAEENTLHLTLAMSLPLNDLPENRTRSELWRRQWLPQKKLSLLLGVNQSVRKAAADCL IDRWFEPYQELLWWEPTDPDGKKLLDKEGRPIKRTAGHMRVLRKLEEIAPFRGYQLGSAV NO:KNGLRHKVADLLLSYAKRKLDPQFTDKTSYPSIGDQFPIVWTGAFVCYEQSITGQLYLY 39LPLFPRGSHQEDITNNYDPDRGPALQVFGEKEIARLSRSTSGLLLPLQFDKWGEATFIRGENNPPTWKATHRRSDKKWLSEVLLREKDFQPKRVELLVRNGRIFVNVACEIPTKPLLEVENFMGVSFGLEHLVTVVVINRDGNVVHQRQEPARRYEKTYFARLERLRRRGGPFSQELETFHYRQVAQIVEEALRFKSVPAVEQVGNIPKGRYNPRLNLRLSYWPFGKLADLTSYKAVKEGLPKPYSVYSATAKMLCSTCGAANKEGDQPISLKGPTVYCGNCGTRHNTGFNTALNLARRAQELFVKGVVAR SEQMSQSLLKWHDMAGRDKDASRSLQKSAVEGVLLHLTASHRVALEMLEKSVSQTVAVT IDMEAAQQRLVIVLEDDPTKATSRKRVISADLQFTREEFGSLPNWAQKLASTCPEIATKYA NO:DKHINSIRIAWGVAKESTNGDAVEQKLQWQIRLLDVTMFLQQLVLQLADKALLEQIPSS 40IRGGIGQEVAQQVTSHIQLLDSGTVLKAELPTISDRNSELARKQWEDAIQTVCTYALPFSRERARILDPGKYAAEDPRGDRLINIDPMWARVLKGPTVKSLPLLFVSGSSIRIVKLTLPRKHAAGHKHTFTATYLVLPVSREWINSLPGTVQEKVQWWKKPDVLATQELLVGKGALKKSANTLVIPISAGKKRFFNHILPALQRGFPLQWQRIVGRSYRRPATHRKWFAQLTIGYTNPSSLPEMALGIHFGMKDILWWALADKQGNILKDGSIPGNSILDFSLQEKGKIERQQKAGKNVAGKKYGKSLLNATYRVVNGVLEFSKGISAEHASQPIGLGLETIRFVDKASGSSPVNARHSNWNYGQLSGIFANKAGPAGFSVTEITLKKAQRDLSDAEQARVLAIEATKRFASRIKRLATKRKDDTLFV SEQVEPVEKERFYYRTYTFRLDGQPRTQNLTTQSGWGLLTKAVLDNTKHYWEIVHHARIAN IDQPIVFENPVIDEQGNPKLNKLGQPRFWKRPISDIVNQLRALFENQNPYQLGSSLIQGTYW NO:DVAENLASWYALNKEYLAGTATWGEPSFPEPHPLTEINQWMPLTFSSGKVVRLLKNAS 41GRYFIGLPILGENNPCYRMRTIEKLIPCDGKGRVTSGSLILFPLVGIYAQQHRRMTDICESIRTEKGKLAWAQVSIDYVREVDKRRRMRRTRKSQGWIQGPWQEVFILRLVLAHKAPKLYKPRCFAGISLGPKTLASCVILDQDERVVEKQQWSGSELLSLIHQGEERLRSLREQSKPTWNAAYRKQLKSLINTQVFTIVTFLRERGAAVRLESIARVRKSTPAPPVNFLLSHWAYRQITERLKDLAIRNGMPLTHSNGSYGVRFTCSQCGATNQGIKDPTKYKVDIESETFLCSICSHREIAAVNTATNLAKQLLDE SEQMNDTETSETLTSHRTVCAHLHVVGETGSLPRLVEAALAELITLNGRATQALLSLAKNGL IDVLRRDKEENLIAAELTLPCRKNKYADVAAKAGEPILATRINNKGKLVTKKWYGEGNSY NO:HIVRFTPETGMFTVRVFDRYAFDEELLHLHSEVVFGSDLPKGIKAKTDSLPANFLQAVFT 42SFLELPFQGFPDIVVKPAMKQAAEQLLSYVQLEAGENQQAEYPDTNERDPELRLVEWQKSLHELSVRTEPFEFVRARDIDYYAETDRRGNRFVNITPEWTKFAESPFARRLPLKIPPEFCILLRRKTEGHAKIPNRIYLGLQIFDGVTPDSTLGVLATAEDGKLFWWHDHLDEFSNLEGKPEPKLKNKPQLLMVSLEYDREQRFEESVGGDRKICLVTLKETRNFRRGWNGRILGIHFQHNPVITWALMDHDAEVLEKGFIEGNAFLGKALDKQALNEYLQKGGKWVGDRSFGNKLKGITHTLASLIVRLAREKDAWIALEEISWVQKQSADSVANHEIVEQPHHSLTR SEQMNDTETSETLTSHRTVCAHLHVVGETGSLPRLVEAALAELITLNGRATQALLSLAKNGL IDVLRRDKEENLIAAELTLPCRKNKYADVAAKAGEPILATRINNKGKLVTKKWYGEGNSY NO:HIVRFTPETGMFTVRVFDRYAFDEELLHLHSEVVFGSDLPKGIKAKTDSLPANFLQAVFT 43SFLELPFQGFPDIVVKPAMKQAAEQLLSYVQLEAGENQQAEYPDTNERDPELRLVEWQKSLHELSVRTEPFEFVRARDIDYYAETDRRGNRFVNITPEWTKFAESPFARRLPLKIPPEFCILLRRKTEGHAKIPNRIYLGLQIFDGVTPDSTLGVLATAEDGKLFWWHDHLDEFSNLEGKPEPKLKNKPQLLMVSLEYDREQRFEESVGGDRKICLVTLKETRNFRRGRHGHTRTDRLPAGNTLWRADFATSAEVAAPKWNGRILGIHFQHNPVITWALMDHDAEVLEKGFIEGNAFLGKALDKQALNEYLQKGGKWVGDRSFGNKLKGITHTLASLIVRLAREKDAWIALEEISWVQKQSADSVANRRFSMWNYSRLATLIEWLGTDIATRDCGTAAPLAHKVSDYLTHFTCPECGACRKAGQKKEIADTVRAGDILTCRKCGFSGPIPDNFIAEFVAKKALERMLKK KPV SEQMAKRNFGEKSEALYRAVRFEVRPSKEELSILLAVSEVLRMLFNSALAERQQVFTEFIASL IDYAELKSASVPEEISEIRKKLREAYKEHSISLFDQINALTARRVEDEAFASVTRNWQEETL NO:DALDGAYKSFLSLRRKGDYDAHSPRSRDSGFFQKIPGRSGFKIGEGRIALSCGAGRKLSF 44PIPDYQQGRLAETTKLKKFELYRDQPNLAKSGRFWISVVYELPKPEATTCQSEQVAFVALGASSIGVVSQRGEEVIALWRSDKHWVPKIEAVEERMKRRVKGSRGWLRLLNSGKRRMHMISSRQHVQDEREIVDYLVRNHGSHFVVTELVVRSKEGKLADSSKPERGGSLGLNWAAQNTGSLSRLVRQLEEKVKEHGGSVRKHKLTLTEAPPARGAENKLWMARKLRESFL KEV SEQLAKNDEKELLYQSVKFEIYPDESKIRVLTRVSNILVLVWNSALGERRARFELYIAPLYEE IDLKKFPRKSAESNALRQKIREGYKEHIPTFFDQLKKLLTPMRKEDPALLGSVPRAYQEETL NO:NTLNGSFVSFMTLRRNNDMDAKPPKGRAEDRFHEISGRSGFKIDGSEFVLSTKEQKLRF 45PIPNYQLEKLKEAKQIKKFTLYQSRDRRFWISIAYEIELPDQRPFNPEEVIYIAFGASSIGVISPEGEKVIDFWRPDKHWKPKIKEVENRMRSCKKGSRAWKKRAAARRKMYAMTQRQQKLNHREIVASLLRLGFHFVVTEYTVRSKPGKLADGSNPKRGGAPQGFNWSAQNTGSFGEFILWLKQKVKEQGGTVQTFRLVLGQSERPEKRGRDNKIEMVRLLREKYLESQTIVV SEQMAKGKKKEGKPLYRAVRFEIFPTSDQITLFLRVSKNLQQVWNEAWQERQSCYEQFFGSI IDYERIGQAKKRAQEAGFSEVWENEAKKGLNKKLRQQEISMQLVSEKESLLQELSIAFQEH NO:GVTLYDQINGLTARRIIGEFALIPRNWQEETLDSLDGSFKSFLALRKNGDPDAKPPRQRV 46SENSFYKIPGRSGFKVSNGQIYLSFGKIGQTLTSVIPEFQLKRLETAIKLKKFELCRDERDMAKPGRFWISVAYEIPKPEKVPVVSKQITYLAIGASRLGVVSPKGEFCLNLPRSDYHWKPQINALQERLEGVVKGSRKWKKRMAACTRMFAKLGHQQKQHGQYEVVKKLLRHGVHFVVTELKVRSKPGALADASKSDRKGSPTGPNWSAQNTGNIARLIQKLTDKASEHGGTVIKRNPPLLSLEERQLPDAQRKIFIAKKLREEFLADQK SEQMAKREKKDDVVLRGTKMRIYPTDRQVTLMDMWRRRCISLWNLLLNLETAAYGAKNT IDRSKLGWRSIWARVVEENHAKALIVYQHGKCKKDGSFVLKRDGTVKHPPRERFPGDRKI NO:LLGLFDALRHTLDKGAKCKCNVNQPYALTRAWLDETGHGARTADIIAWLKDFKGECD 47CTAISTAAKYCPAPPTAELLTKIKRAAPADDLPVDQAILLDLFGALRGGLKQKECDHTHARTVAYFEKHELAGRAEDILAWLIAHGGTCDCKIVEEAANHCPGPRLFIWEHELAMIMARLKAEPRTEWIGDLPSHAAQTVVKDLVKALQTMLKERAKAAAGDESARKTGFPKFKKQAYAAGSVYFPNTTMFFDVAAGRVQLPNGCGSMRCEIPRQLVAELLERNLKPGLVIGAQLGLLGGRIWRQGDRWYLSCQWERPQPTLLPKTGRTAGVKIAASIVFTTYDNRGQTKEYPMPPADKKLTAVHLVAGKQNSRALEAQKEKEKKLKARKERLRLGKLEKGHDPNALKPLKRPRVRRSKLFYKSAARLAACEAIERDRRDGFLHRVTNEIVHKFDAVSVQKMSVAPMMRRQKQKEKQIESKKNEAKKEDNGAAKKPRNLKPVRKLLRHVAMARGRQFLEYKYNDLRGPGSVLIADRLEPEVQECSRCGTKNPQMKDGRRLLRCIGVLPDGTDCDAVLPRNRNAARNAEKRLRKHREAHNA SEQMNEVLPIPAVGEDAADTIMRGSKMRIYPSVRQAATMDLWRRRCIQLWNLLLELEQAAY IDSGENRRTQIGWRSIWATVVEDSHAEAVRVAREGKKRKDGTFRKAPSGKEIPPLDPAML NO:AKIQRQMNGAVDVDPKTGEVTPAQPRLFMWEHELQKIMARLKQAPRTHWIDDLPSHA 48AQSVVKDLIKALQAMLRERKKRASGIGGRDTGFPKFKKNRYAAGSVYFANTQLRFEAKRGKAGDPDAVRGEFARVKLPNGVGWMECRMPRHINAAHAYAQATLMGGRIWRQGENWYLSCQWKMPKPAPLPRAGRTAAIKIAAAIPITTVDNRGQTREYAMPPIDRERIAAHAAAGRAQSRALEARKRRAKKREAYAKKRHAKKLERGIAAKPPGRARIKLSPGFYAAAAKLAKLEAEDANAREAWLHEITTQIVRNFDVIAVPRMEVAKLMKKPEPPEEKEEQVKAPWQGKRRSLKAARVMMRRTAMALIQTTLKYKAVDLRGPQAYEEIAPLDVTAAACSGCGVLKPEWKMARAKGREIMRCQEPLPGGKTCNTVLTYTRNSARVIGRELAVRLAERQKA SEQMTTQKTYNFCFYDQRFFELSKEAGEVYSRSLEEFWKIYDETGVWLSKFDLQKHMRNKL IDERKLLHSDSFLGAMQQVHANLASWKQAKKVVPDACPPRKPKFLQAILFKKSQIKYKNG NO:FLRLTLGTEKEFLYLKWDINIPLPIYGSVTYSKTRGWKINLCLETEVEQKNLSENKYLSID 49LGVKRVATIFDGENTITLSGKKFMGLMHYRNKLNGKTQSRLSHKKKGSNNYKKIQRAKRKTTDRLLNIQKEMLHKYSSFIVNYAIRNDIGNIIIGDNSSTHDSPNMRGKTNQKISQNPEQKLKNYIKYKFESISGRVDIVPEPYTSRKCPHCKNIKKSSPKGRTYKCKKCGFIFDRDGVGAINIYNENVSFGQIISPGRIRSLTEPIGMKFHNEIYFKSYVAA SEQMSVRSFQARVECDKQTMEHLWRTHKVFNERLPEIIKILFKMKRGECGQNDKQKSLYKS IDISQSILEANAQNADYLLNSVSIKGWKPGTAKKYRNASFTWADDAAKLSSQGIHVYDKK NO:QVLGDLPGMMSQMVCRQSVEAISGHIELTKKWEKEHNEWLKEKEKWESEDEHKKYL 50DLREKFEQFEQSIGGKITKRRGRWHLYLKWLSDNPDFAAWRGNKAVINPLSEKAQIRINKAKPNKKNSVERDEFFKANPEMKALDNLHGYYERNFVRRRKTKKNPDGFDHKPTFTLPHPTIHPRWFVFNKPKTNPEGYRKLILPKKAGDLGSLEMRLLTGEKNKGNYPDDWISVKFKADPRLSLIRPVKGRRVVRKGKEQGQTKETDSYEFFDKHLKKWRPAKLSGVKLIFPDKTPKAAYLYFTCDIPDEPLTETAKKIQWLETGDVTKKGKKRKKKVLPHGLVSCAVDLSMRRGTTGFATLCRYENGKIHILRSRNLWVGYKEGKGCHPYRWTEGPDLGHIAKHKREIRILRSKRGKPVKGEESHIDLQKHIDYMGEDRFKKAARTIVNFALNTENAASKNGFYPRADVLLLENLEGLIPDAEKERGINRALAGWNRRHLVERVIEMAKDAGFKRRVFEIPPYGTSQVCSKCGALGRRYSIIRENNRREIRFGYVEKLFACPNCGYCANADHNASVNLNRRFLIEDSFKSYYDWKRLSEKKQKEEIETIESKLMDKLCAMHKISRGSISK SEQMHLWRTHCVFNQRLPALLKRLFAMRRGEVGGNEAQRQVYQRVAQFVLARDAKDSVD IDLLNAVSLRKRSANSAFKKKATISCNGQAREVTGEEVFAEAVALASKGVFAYDKDDMR NO:AGLPDSLFQPLTRDAVACMRSHEELVATWKKEYREWRDRKSEWEAEPEHALYLNLRP 51KFEEGEAARGGRFRKRAERDHAYLDWLEANPQLAAWRRKAPPAVVPIDEAGKRRIARAKAWKQASVRAEEFWKRNPELHALHKIHVQYLREFVRPRRTRRNKRREGFKQRPTFTMPDPVRHPRWCLFNAPQTSPQGYRLLRLPQSRRTVGSVELRLLTGPSDGAGFPDAWVNVRFKADPRLAQLRPVKVPRTVTRGKNKGAKVEADGFRYYDDQLLIERDAQVSGVKLLFRDIRMAPFADKPIEDRLLSATPYLVFAVEIKDEARTERAKAIRFDETSELTKSGKKRKTLPAGLVSVAVDLDTRGVGFLTRAVIGVPEIQQTHHGVRLLQSRYVAVGQVEARASGEAEWSPGPDLAHIARHKREIRRLRQLRGKPVKGERSHVRLQAHIDRMGEDRFKKAARKIVNEALRGSNPAAGDPYIRADVLLYESLETLLPDAERERGINRALLRWNRAKLIEHLKRMCDDAGIRHFPVSPFGTSQVCSKCGALGRRYSLARENGRAVIRFGWVERLFACPNPECPGRRPDRPDRPFTCNSDHNASVNLHRVFALGDQAVAAFRALAPRDSPARTLAVKRVEDTLRPQLMRVHKLADAGVDSPF SEQMATLVYRYGVRAHGSARQQDAVVSDPAMLEQLRLGHELRNALVGVQHRYEDGKRAV IDWSGFASVAAADHRVTTGETAVAELEKQARAEHSADRTAATRQGTAESLKAARAAVK NO:QARADRKAAMAAVAEQAKPKIQALGDDRDAEIKDLYRRFCQDGVLLPRCGRCAGDLR 52SDGDCTDCGAAHEPRKLYWATYNAIREDHQTAVKLVEAKRKAGQPARLRFRRWTGDGTLTVQLQRMHGPACRCVTCAEKLTRRARKTDPQAPAVAADPAYPPTDPPRDPALLASGQGKWRNVLQLGTWIPPGEWSAMSRAERRRVGRSHIGWQLGGGRQLTLPVQLHRQMPADADVAMAQLTRVRVGGRHRMSVALTAKLPDPPQVQGLPPVALHLGWRQRPDGSLRVATWACPQPLDLPPAVADVVVSHGGRWGEVIMPARWLADAEVPPRLLGRRDKAMEPVLEALADWLEAHTEACTARMTPALVRRWRSQGRLAGLTNRWRGQPPTGSAEILTYLEAWRIQDKLLWERESHLRRRLAARRDDAWRRVASWLARHAGVLVVDDADIAELRRRDDPADTDPTMPASAAQAARARAALAAPGRLRHLATITATRDGLGVHTVASAGLTRLHRKCGHQAQPDPRYAASAVVTCPGCGNGYDQDYNAAMLMLDRQQQP SEQMSRVELHRAYKFRLYPTPAQVAELAEWERQLRRLYNLAHSQRLAAMQRHVRPKSPGV IDLKSECLSCGAVAVAEIGTDGKAKKTVKHAVGCSVLECRSCGGSPDAEGRTAHTAACSF NO:VDYYRQGREMTQLLEEDDQLARVVCSARQETLRDLEKAWQRWHKMPGFGKPHFKKR 53IDSCRIYFSTPKSWAVDLGYLSFTGVASSVGRIKIRQDRVWPGDAKFSSCHVVRDVDEWYAVFPLTFTKEIEKPKGGAVGINRGAVHAIADSTGRVVDSPKFYARSLGVIRFIRARLLDRKVPFGRAVKPSPTKYHGLPKADIDAAAARVNASPGRLVYEARARGSIAAAEAHLAALVLPAPRQTSQLPSEGRNRERARRFLALAHQRVRRQREWFLHNESAHYAQSYTKIAIEDWSTKEMTSSEPRDAEEMKRVTRARNRSILDVGWYELGRQIAYKSEATGAEFAKVDPGLRETETHVPEAIVRERDVDVSGMLRGEAGISGTCSRCGGLLRASASGHADAECEVCLHVEVGDVNAAVNVLKRAMFPGAAPPSKEKAKVTIGIKGRKKKRAA SEQMSRVELHRAYKFRLYPTPVQVAELSEWERQLRRLYNLGHEQRLLTLTRHLRPKSPGVL IDKGECLSCDSTQVQEVGADGRPKTTVRHAEQCPTLACRSCGALRDAEGRTAHTVACAF NO:VDYYRQGREMTELLAADDQLARVVCSARQEVLRDLDKAWQRWRKMPGFGKPRFKRR 54TDSCRIYFSTPKAWKLEGGHLSFTGAATTVGAIKMRQDRNWPASVQFSSCHVVRDVDEWYAVFPLTFVAEVARPKGGAVGINRGAVHAIADSTGRVVDSPRYYARALGVIRHRARLFDRKVPSGHAVKPSPTKYRGLSAIEVDRVARATGFTPGRVVTEALNRGGVAYAECALAAIAVLGHGPERPLTSDGRNREKARKFLALAHQRVRRQREWFLHNESAHYARTYSKIAIEDWSTKEMTASEPQGEETRRVTRSRNRSILDVGWYELGRQLAYKTEATGAEFAQVDPGLKETETNVPKAIADARDVDVSGMLRGEAGISGTCSKCGGLLRAPASGHADAECEICLNVEVGDVNAAVNVLKRAMFPGDAPPASGEKPKVSIGIKGRQKKKKAA SEQMEAIATGMSPERRVELGILPGSVELKRAYKFRLYPMKVQQAELSEWERQLRRLYNLAH IDEQRLAALLRYRDWDFQKGACPSCRVAVPGVHTAACDHVDYFRQAREMTQLLEVDAQ NO:LSRVICCARQEVLRDLDKAWQRWRKKLGGRPRFKRRTDSCRIYLSTPKHWEIAGRYLR 55LSGLASSVGEIRIEQDRAFPEGALLSSCSIVRDVDEWYACLPLTFTQPIERAPHRSVGLNRGVVHALADSDGRVVDSPKFFERALATVQKRSRDLARKVSGSRNAHKARIKLAKAHQRVRRQRAAFLHQESAYYSKGFDLVALEDMSVRKMTATAGEAPEMGRGAQRDLNRGILDVGWYELARQIDYKRLAHGGELLRVDPGQTTPLACVTEEQPARGISSACAVCGIPLARPASGNARMRCTACGSSQVGDVNAAENVLTRALSSAPSGPKSPKASIKIKGRQKRLGTPANRAGEASGGDPPVRGPVEGGTLAYVVEPVSESQSDT SEQMTVRTYKYRAYPTPEQAEALTSWLRFASQLYNAALEHRKNAWGRHDAHGRGFRFWD IDGDAAPRKKSDPPGRWVYRGGGGAHISKNDQGKLLTEFRREHAELLPPGMPALVQHEV NO:LARLERSMAAFFQRATKGQKAGYPRWRSEHRYDSLTFGLTSPSKERFDPETGESLGRG 56KTVGAGTYHNGDLRLTGLGELRILEHRRIPMGAIPKSVIVRRSGKRWFVSIAMEMPSVEPAASGRPAVGLDMGVVTWGTAFTADTSAAAALVADLRRMATDPSDCRRLEELEREAAQLSEVLAHCRARGLDPARPRRCPKELTKLYRRSLHRLGELDRACARIRRRLQAAHDIAEPVPDEAGSAVLIEGSNAGMRHARRVARTQRRVARRTRAGHAHSNRRKKAVQAYARAKERERSARGDHRHKVSRALVRQFEEISVEALDIKQLTVAPEHNPDPQPDLPAHVQRRRNRGELDAAWGAFFAALDYKAADAGGRVARKPAPHTTQECARCGTLVPKPISLRVFIRCPACGYTAPRTVNSARNVLQRPLEEPGRAGPSGANGRGVPHAVA SEQMNCRYRYRIYPTPGQRQSLARLFGCVRVVWNDALFLCRQSEKLPKNSELQKLCITQAK IDKTEARGWLGQVSAIPLQQSVADLGVAFKNFFQSRSGKRKGKKVNPPRVKRRNNRQGA NO:RFTRGGFKVKTSKVYLARIGDIKIKWSRPLPSEPSSVTVIKDCAGQYFLSFVVEVKPEIKP 57PKNPSIGIDLGLKTFASCSNGEKIDSPDYSRLYRKLKRCQRRLAKRQRGSKRRERMRVKVAKLNAQIRDKRKDFLHKLSTKVVNENQVIALEDLNVGGMLKNRKLSRAISQAGWYEFRSLCEGKAEKHNRDFRVISRWEPTSQVCSECGYRWGKIDLSVRSIVCINCGVEHDRDDNASVNIEQAGLKVGVGHTHDSKRTGSACKTSNGAVCVEPSTHREYVQLTLFDW SEQMKSRWTFRCYPTPEQEQHLARTFGCVRFVWNWALRARTDAFRAGERIGYPATDKALT IDLLKQQPETVWLNEVSSVCLQQALRDLQVAFSNFFDKRAAHPSFKRKEARQSANYTERG NO:FSFDHERRILKLAKIGAIKVKWSRKAIPHPSSIRLIRTASGKYFVSLVVETQPAPMPETGE 58SVGVDFGVARLATLSNGERISNPKHGAKWQRRLAFYQKRLARATKGSKRRMRIKRHVARIHEKIGNSRSDTLHKLSTDLVTRFDLICVEDLNLRGMVKNHSLARSLHDASIGSAIRMIEEKAERYGKNVVKIDRWFPSSKTCSDCGHIVEQLPLNVREWTCPECGTTHDRDANAAANILAVGQTVSAHGGTVRRSRAKASERKSQRSANRQGVNRA SEQKEPLNIGKTAKAVFKEIDPTSLNRAANYDASIELNCKECKFKPFKNVKRYEFNFYNWY IDRCNPNSCLQSTYKAQVRKVEIGYEKLKNEILTQMQYYPWFGRLYQNFFHDERDKMTSL NO:DEIQVIGVQNKVFFNTVEKAWREIIKKRFKDNKETMETIPELKHAAGHGKRKLSNKSLL 59RRRFAFVQKSFKFVDNSDVSYRSFSNNIACVLPSRIGVDLGGVISRNPKREYIPQEISFNAFWKQHEGLKKGRNIEIQSVQYKGETVKRIEADTGEDKAWGKNRQRRFTSLILKLVPKQGGKKVWKYPEKRNEGNYEYFPIPIEFILDSGETSIRFGGDEGEAGKQKHLVIPFNDSKATPLASQQTLLENSRFNAEVKSCIGLAIYANYFYGYARNYVISSIYHKNSKNGQAITAIYLESIAHNYVKAIERQLQNLLLNLRDFSFMESHKKELKKYFGGDLEGTGGAQKRREKEEKIEKEIEQSYLPRLIRLSLTKMVTKQVEM SEQELIVNENKDPLNIGKTAKAVFKEIDPTSINRAANYDASIELACKECKFKPFNNTKRHDFS IDFYSNWHRCSPNSCLQSTYRAKIRKTEIGYEKLKNEILNQMQYYPWFGRLYQNFFNDQR NO:DKMTSLDEIQVTGVQNKIFFNTVEKAWREIIKKRFRDNKETMRTIPDLKNKSGHGSRKL 60SNKSLLRRRFAFAQKSFKLVDNSDVSYRAFSNNVACVLPSKIGVDIGGIINKDLKREYIPQEITFNVFWKQHDGLKKGRNIEIHSVQYKGEIVKRIEADTGEDKAWGKNRQRRFTSLILKITPKQGGKKIWKFPEKKNASDYEYFPIPIEFILDNGDASIKFGGEEGEVGKQKHLLIPFNDSKATPLSSKQMLLETSRFNAEVKSTIGLALYANYFVSYARNYVIKSTYHKNSKKGQIVTEIYLESISQNFVRAIQRQLQSLMLNLKDWGFMQTHKKELKKYFGSDLEGSKGGQKRREKEEKIEKEIEASYLPRLIRLSLTKSVTKAEEM SEQPEEKTSKLKPNSINLAANYDANEKFNCKECKFHPFKNKKRYEFNFYNNLHGCKSCTKST IDNNPAVKRIEIGYQKLKFEIKNQMEAYPWFGRLRINFYSDEKRKMSELNEMQVTGVKNK NO:IFFDAIECAWREILKKRFRESKETLITIPKLKNKAGHGARKHRNKKLLIRRRAFMKKNFH 61FLDNDSISYRSFANNIACVLPSKVGVDIGGIISPDVGKDIKPVDISLNLMWASKEGIKSGRKVEIYSTQYDGNMVKKIEAETGEDKSWGKNRKRRQTSLLLSIPKPSKQVQEFDFKEWPRYKDIEKKVQWRGFPIKIIFDSNHNSIEFGTYQGGKQKVLPIPFNDSKTTPLGSKMNKLEKLRFNSKIKSRLGSAIAANKFLEAARTYCVDSLYHEVSSANAIGKGKIFIEYYLEILSQNYIEAAQKQLQRFIESIEQWFVADPFQGRLKQYFKDDLKRAKCFLCANREVQTTCYAAVKLHKSCAEKVKDKNKELAIKERNNKEDAVIKEVEASNYPRVIRLKLTKTITNKAM SEQSESENKIIEQYYAFLYSFRDKYEKPEFKNRGDIKRKLQNKWEDFLKEQNLKNDKKLSNY IDIFSNRNFRRSYDREEENEEGIDEKKSKPKRINCFEKEKNLKDQYDKDAINASANKDGAQ NO:KWGCFECIFFPMYKIESGDPNKRIIINKTRFKLFDFYLNLKGCKSCLRSTYHPYRSNVYIE 62SNYDKLKREIGNFLQQKNIFQRMRKAKVSEGKYLTNLDEYRLSCVAMHFKNRWLFFDSIQKVLRETIKQRLKQMRESYDEQAKTKRSKGHGRAKYEDQVRMIRRRAYSAQAHKLLDNGYITLFDYDDKEINKVCLTAINQEGFDIGGYLNSDIDNVMPPIEISFHLKWKYNEPILNIESPFSKAKISDYLRKIREDLNLERGKEGKARSKKNVRRKVLASKGEDGYKKIFTDFFSKWKEELEGNAMERVLSQSSGDIQWSKKKRIHYTTLVLNINLLDKKGVGNLKYYEIAEKTKILSFDKNENKFWPITIQVUDGYEIGTEYDEIKQLNEKTSKQFTIYDPNTKIIKIPFTDSKAVPLGMLGINIATLKTVKKTERDIKVSKIFKGGLNSKIVSKIGKGIYAGYFPTVDKEILEEVEEDTLDNEFSSKSQRNIFLKSIIKNYDKMLKEQLFDFYSFLVRNDLGVRFLTDRELQNIEDESFNLEKRFFETDRDRIARWFDNTNTDDGKEKFKKLANEIVDSYKPRLIRLPVVRVIK RIQPVKQREMSEQ KYSTRDFSELNEIQVTACKQDEFFKVIQNAWREIIKKRFLENRENFIEKKIFKNKKGRGK IDRQESDKTIQRNRASVMKNFQLIENEKIILRAPSGHVACVFPVKVGLDIGGFKTDDLEKNI NO:FPPRTITINVFWKNRDRQRKGRKLEVWGIKARTKLIEKVHKWDKLEEVKKKRLKSLEQ 63KQEKSLDNWSEVNNDSFYKVQIDELQEKIDKSLKGRTMNKILDNKAKESKEAEGLYIEWEKDFEGEMLRRIEASTGGEEKWGKRRQRRHTSLLLDIKNNSRGSKEIINFYSYAKQGKKEKKIEFFPFPLTITLDAEEESPLNIKSIPIEDKNATSKYFSIPFTETRATPLSILGDRVQKFKTKNISGAIKRNLGSSISSCKIVQNAETSAKSILSLPNVKEDNNMEIFINTMSKNYFRAMMKQMESFIFEMEPKTLIDPYKEKAIKWFEVAASSRAKRKLKKLSKADIKKSELLLSNTEEFEKEKQEKLEALEKEIEEFYLPRIVRLQLTKTILETPVM SEQKKLQLLGHKILLKEYDPNAVNAAANFETSTAELCGQCKMKPFKNKRRFQYTFGKNYH IDGCLSCIQNVYYAKKRIVQIAKEELKHQLTDSIASIPYKYTSLFSNTNSIDELYILKQERAA NO:FFSNTNSIDELYITGIENNIAFKVISAIWDEIIKKRRQRYAESLTDTGTVKANRGHGGTAY 64KSNTRQEKIRALQKQTLHMVTNPYISLARYKNNYIVATLPRTIGMHIGAIKDRDPQKKLSDYAINFNVFWSDDRQLIELSTVQYTGDMVRKIEAETGENNKWGENMKRTKTSLLLEILTKKTTDELTFKDWAFSTKKEIDSVTKKTYQGFPIGIIFEGNESSVKFGSQNYFPLPFDAKITPPTAEGFRLDWLRKGSFSSQMKTSYGLAIYSNKVTNAIPAYVIKNMFYKIARAENGKQIKAKFLKKYLDIAGNNYVPFIIMQHYRVLDTFEEMPISQPKVIRLSLTKTQHIIIKKDKTDS KM SEQNTSNLINLGKKAINISANYDANLEVGCKNCKFLSSNGNFPRQTNVKEGCHSCEKSTYEP IDSIYLVKIGERKAKYDVLDSLKKFTFQSLKYQSKKSMKSRNKKPKELKEFVIFANKNKAF NO:DVIQKSYNHLILQIKKEINRMNSKKRKKNHKRRLFRDREKQLNKLRLIESSNLFLPRENK 65GNNHVFTYVAIHSVGRDIGVIGSYDEKLNFETELTYQLYFNDDKRLLYAYKPKQNKIIKIKEKLWNLRKEKEPLDLEYEKPLNKSITFSIKNDNLFKVSKDLMLRRAKFNIQGKEKLSKEERKINRDLIKIKGLVNSMSYGRFDELKKEKNIWSPHIYREVRQKEIKPCLIKNGDRIEIFEQLKKKMERLRRFREKRQKKISKDLIFAERIAYNFHTKSIKNTSNKINIDQEAKRGKASYMRKRIGYETFKNKYCEQCLSKGNVYRNVQKGCSCFENPFDWIKKGDENLLPKKNEDLRVKGAFRDEALEKQIVKIAFNIAKGYEDFYDNLGESTEKDLKLKFKVGTTINEQESLKL SEQTSNPIKLGKKAINISANYDSNLQIGCKNCKFLSYNGNFPRQTNVKEGCHSCEKSTYEPPV IDYTVRIGERRSKYDVLDSLKKFIFLSLKYRQSKKMKTRSKGIRGLEEFVISANLKKAMDVI NO:QKSYRHLILNIKNEIVRMNGKKRNKNHKRLLFRDREKQLNKLRLIEGSSFFKPPTVKGD 66NSIFTCVAIHNIGRDIGIAGDYFDKLEPKIELTYQLYYEYNPKKESEINKRLLYAYKPKQNKIIEIKEKLWNLRKEKSPLDLEYEKPLTKSITFLVKRDGVFRISKDLMLRKAKFIIQGKEKLSKEERKINRDLIKIKSNIISLTYGRFDELKKDKTIWSPHIFRDVKQGKITPCIERKGDRMDIFQQLRKKSERLRENRKKRQKKISKDLIFAERIAYNFHTKSIKNTSNLINIKHEAKRGKASYMRKRIGNETFRIKYCEQCFPKNNVYKNVQKGCSCFEDPFEYIKKGNEDLIPNKNQDLKAKGAFRDDALEKQIIKVAFNIAKGYEDFYENLKKTTEKDIRLKFKVGTIISEEM SEQNNSINLSKKAINISANYDANLQVRCKNCKFLSSNGNFPRQTDVKEGCHSCEKSTYEPPV IDYDVKIGEIKAKYEVLDSLKKFTFQSLKYQLSKSMKFRSKKIKELKEFVIFAKESKALNVI NO:NRSYKHLILNIKNDINRMNSKKRIKNHKGRLFLDRQKQLSKLKLIEGSSFFVPAKNVGN 67KSVFTCVAIHSIGRDIGIAGLYDSFTKPVNEITYQIFFSGERRLLYAYKPKQLKILSIKENLWSLKNEKKPLDLLYEKPLGKNLNFNVKGGDLFRVSKDLMIRNAKFNVHGRQRLSDEERLINRNFIKIKGEVVSLSYGRFEELKKDRKLWSPHIFKDVRQNKIKPCLVMQGQRIDIFEQLKRKLELLKKIRKSRQKKLSKDLIFGERIAYNFHTKSIKNTSNKINIDSDAKRGRASYMRKRIGNETFKLKYCDVCFPKANVYRRVQNGCSCSENPYNYIKKGDKDLLPKKDEGLAIKGAFRDEKLNKQIIKVAFNIAKGYEDFYDDLKKRTEKDVDLKFKIGTTVLDQKPMEIFD GIVITWL SEQLLTTVVETNNLAKKAINVAANFDANIDRQYYRCTPNLCRFIAQSPRETKEKDAGCSSCT IDQSTYDPKVYVIKIGKLLAKYEILKSLKRFLFMNRYFKQKKTERAQQKQKIGTELNEMSIF NO:AKATNAMEVIKRATKHCTYDIIPETKSLQMLKRRRHRVKVRSLLKILKERRMKIKKIPN 68TFIEIPKQAKKNKSDYYVAAALKSCGIDVGLCGAYEKNAEVEAEYTYQLYYEYKGNSSTKRILYCYNNPQKNIREFWEAFYIQGSKSHVNTPGTIRLKMEKFLSPITIESEALDFRVWNSDLKIRNGQYGFIKKRSLGKEAREIKKGMGDIKRKIGNLTYGKSPSELKSIHVYRTERENPKKPRAARKKEDNFMEIFEMQRKKDYEVNKKRRKEATDAAKIMDFAEEPIRHYHTNNLKAVRRIDMNEQVERKKTSVFLKRIMQNGYRGNYCRKCIKAPEGSNRDENVLEKNEGCLDCIGSEFIWKKSSKEKKGLWHTNRLLRRIRLQCFTTAKAYENFYNDLFEKKESSLDIIKL KVSITTKSMSEQ ASTMNLAKQAINFAANYDSNLEIGCKGCKFMSTWSKKSNPKFYPRQNNQANKCHSCT IDYSTGEPEVPIIEIGERAAKYKIFTALKKFVFMSVAYKERRRQRFKSKKPKELKELAICSNR NO:EKAMEVIQKSVVHCYGDVKQEIPRIRKIKVLKNHKGRLFYKQKRSKIKIAKLEKGSFFK 69TFIPKVHNNGCHSCHEASLNKPILVTTALNTIGADIGLINDYSTIAPTETDISWQVYYEFIPNGDSEAVKKRLLYFYKPKGALIKSIRDKYFKKGHENAVNTGFFKYQGKIVKGPIKFVNNELDFARKPDLKSMKIKRAGFAIPSAKRLSKEDREINRESIKIKNKIYSLSYGRKKTLSDKDIIKHLYRPVRQKGVKPLEYRKAPDGFLEFFYSLKRKERRLRKQKEKRQKDMSEIIDAADEFAWHRHTGSIKKTTNHINFKSEVKRGKVPIMKKRIANDSFNTRHCGKCVKQGNAINKYYIEKQKNCFDCNSIEFKWEKAALEKKGAFKLNKRLQYIVKACFNVAKAYESFYEDFRKGEEESLDLKFKIGTTTTLKQYPQNKARAM SEQHSHNLMLTKLGKQAINFAANYDANLEIGCKNCKFLSYSPKQANPKKYPRQTDVHEDGN IDIACHSCMQSTKEPPVYIVPIGERKSKYEILTSLNKFTFLALKYKEKKRQAFRAKKPKELQ NO:ELAIAFNKEKAIKVIDKSIQHLILNIKPEIARIQRQKRLKNRKGKLLYLHKRYAIKMGLIK 70NGKYFKVGSPKKDGKKLLVLCALNTIGRDIGIIGNIEENNRSETEITYQLYFDCLDANPNELRIKEIEYNRLKSYERKIKRLVYAYKPKQTKILEIRSKFFSKGHENKVNTGSFNFENPLNKSISIKVKNSAFDFKIGAPFIMLRNGKFHIPTKKRLSKEEREINRTLSKIKGRVFRLTYGRNISEQGSKSLHIYRKERQHPKLSLEIRKQPDSFIDEFEKLRLKQNFISKLKKQRQKKLADLLQFADRIAYNYHTSSLEKTSNFINYKPEVKRGRTSYIKKRIGNEGFEKLYCETCIKSNDKENAYAVEKEELCFVCKAKPFTWKKTNKDKLGIFKYPSRIKDFIRAAFTVAKSYNDFYENLKKKDLKNEIFLKFKIGLILSHEKKNHISIAKSVAEDERISGKSIKNILNKSIKLEKNCYSCFF HKEDMSEQ SLERVIDKRNLAKKAINIAANFDANINKGFYRCETNQCMFIAQKPRKTNNTGCSSCLQST IDYDPVIYVVKVGEMLAKYEILKSLKRFVFMNRSFKQKKTEKAKQKERIGGELNEMSIFA NO:NAALAMGVIKRAIRHCHVDIRPEINRLSELKKTKHRVAAKSLVKIVKQRKTKWKGIPNS 71FIQIPQKARNKDADFYVASALKSGGIDIGLCGTYDKKPHADPRWTYQLYFDTEDESEKRLLYCYNDPQAKIRDFWKTFYERGNPSMVNSPGTIEFRMEGFFEKMTPISIESKDFDFRVWNKDLLIRRGLYEIKKRKNLNRKAREIKKAMGSVKRVLANMTYGKSPTDKKSIPVYRVEREKPKKPRAVRKEENELADKLENYRREDFLIRNRRKREATEIAKIIDAAEPPIRHYHTNHLRAVKRIDLSKPVARKNTSVFLKRIMQNGYRGNYCKKCIKGNIDPNKDECRLEDIKKCICCEGTQNIWAKKEKLYTGRINVLNKRIKQMKLECFNVAKAYENFYDNLAALKEGDLKVLKLKVSIPALNPEASDPEEDM SEQNASINLGKRAINLSANYDSNLVIGCKNCKFLSFNGNFPRQTNVREGCHSCDKSTYAPEV IDYIVKIGERKAKYDVLDSLKKFTFQSLKYQIKKSMRERSKKPKELLEFVIFANKDKAFNVI NO:QKSYEHLILNIKQEINRMNGKKRIKNHKKRLFKDREKQLNKLRLIGSSSLFFPRENKGDK 405DLFTYVAIHSVGRDIGVAGSYESHIEPISDLTYQLFINNEKRLLYAYKPKQNKIIELKENLWNLKKEKKPLDLEFTKPLEKSITFSVKNDKLFKVSKDLMLRQAKFNIQGKEKLSKEERQINRDFSKIKSNVISLSYGRFEELKKEKNIWSPHIYREVKQKEIKPCIVRKGDRIELFEQLKRKMDKLKKFRKERQKKISKDLNFAERIAYNFHTKSIKNTSNKINIDQEAKRGKASYMRKRIGNESFRKKYCEQCFSVGNVYHNVQNGCSCFDNPIELIKKGDEGLIPKGKEDRKYKGALRDDNLQMQIIRVAFNIAKGYEDFYNNLKEKTEKDLKLKFKIGTTISTQESNNKEM SEQSNLIKLGKQAINFAANYDANLEVGCKNCKFLSSTNKYPRQTNVHLDNKMACRSCNQST IDMEPAIYIVRIGEKKAKYDIYNSLTKFNFQSLKYKAKRSQRFKPKQPKELQELSIAVRKEK NO:ALDIIQKSIDHLIQDIRPEIPRIKQQKRYKNHVGKLFYLQKRRKNKLNLIGKGSFFKVFSP 72KEKKNELLVICALTNIGRDIGLIGNYNTIINPLFEVTYQLYYDYIPKKNNKNVQRRLLYAYKSKNEKILKLKEAFFKRGHENAVNLGSFSYEKPLEKSLTLKIKNDKDDFQVSPSLRIRTGRFFVPSKRNLSRQEREINRRLVKIKSKIKNMTYGKFETARDKQSVHIFRLERQKEKLPLQFRKDEKEFMEEFQKLKRRTNSLKKLRKSRQKKLADLLQLSEKVVYNNHTGTLKKTSNFLNFSSSVKRGKTAYIKELLGQEGFETLYCSNCINKGQKTRYNIETKEKCFSCKDVPFVWKKKSTDKDRKGAFLFPAKLKDVIKATFTVAKAYEDFYDNLKSIDEKKPYIKFKIGLILAHVRHEHKARAKEEAGQKNIYNKPIKIDKNCKECFFFKEEAM SEQNTTRKKFRKRTGFPQSDNIKLAYCSAIVRAANLDADIQKKHNQCNPNLCVGIKSNEQSR IDKYEHSDRQALLCYACNQSTGAPKVDYIQIGEIGAKYKILQMVNAYDFLSLAYNLTKLR NO:NGKSRGHQRMSQLDEVVIVADYEKATEVIKRSINHLLDDIRGQLSKLKKRTQNEHITEH 73KQSKIRRKLRKLSRLLKRRRWKWGTIPNPYLKNWVFTKKDPELVTVALLHKLGRDIGLVNRSKRRSKQKLLPKVGFQLYYKWESPSLNNIKKSKAKKLPKRLLIPYKNVKLFDNKQKLENAIKSLLESYQKTIKVEFDQFFQNRTEEIIAEEQQTLERGLLKQLEKKKNEFASQKKALKEEKKKIKEPRKAKLLMEESRSLGFLMANVSYALFNTTIEDLYKKSNVVSGCIPQEPVVVFPADIQNKGSLAKILFAPKDGFRIKFSGQHLTIRTAKFKIRGKEIKILTKTKREILKNIEKLRRVWYREQHYKLKLFGKEVSAKPRFLDKRKTSIERRDPNKLADQTDDRQAELRNKEYELRHKQHKMAERLDNIDTNAQNLQTLSFWVGEADKPPKLDEKDARGFGVRTCISAWKWFMEDLLKKQEEDPLLKLKLSIM SEQPKKPKFQKRTGFPQPDNLRKEYCLAIVRAANLDADFEKKCTKCEGIKTNKKGNIVKGRT IDYNSADKDNLLCYACNISTGAPAVDYVFVGALEAKYKILQMVKAYDFHSLAYNLAKLW NO:KGRGRGHQRMGGLNEVVIVSNNEKALDVIEKSLNHFHDEIRGELSRLKAKFQNEHLHV 74HKESKLRRKLRKISRLLKRRRWKWDVIPNSYLRNFTFTKTRPDFISVALLHRVGRDIGLVTKTKIPKPTDLLPQFGFQIYYTWDEPKLNKLKKSRLRSEPKRLLVPYKKIELYKNKSVLEEAIRHLAEVYTEDLTICFKDFFETQKRKFVSKEKESLKRELLKELTKLKKDFSERKTALKRDRKEIKEPKKAKLLMEESRSLGFLAANTSYALFNLIAADLYTKSKKACSTKLPRQLSTILPLEIKEHKSTTSLAIKPEEGFKIRFSNTHLSIRTPKFKMKGADIKALTKRKREILKNATKLEKSWYGLKHYKLKLYGKEVAAKPRFLDKRNPSIDRRDPKELMEQIENRRNEVKDLEYEIRKGQHQMAKRLDNVDTNAQNLQTKSFWVGEADKPPELDSMEAKKLGLRTCISAWKWFMKDLVLLQEKSPNLKLKLSLTEM SEQKFSKRQEGFLIPDNIDLYKCLAIVRSANLDADVQGHKSCYGVKKNGTYRVKQNGKKGV IDKEKGRKYVFDLIAFKGNIEKIPHEAIEEKDQGRVIVLGKFNYKLILNIEKNHNDRASLEIK NO:NKIKKLVQISSLETGEFLSDLLSGKIGIDEVYGIIEPDVFSGKELVCKACQQSTYAPLVEY 75MPVGELDAKYKILSAIKGYDFLSLAYNLSRNRANKKRGHQKLGGGELSEVVISANYDKALNVIKRSINHYHVEIKPEISKLKKKMQNEPLKVMKQARIRRELHQLSRKVKRLKWKWGMIPNPELQNIIFEKKEKDFVSYALLHTLGRDIGLFKDTSMLQVPNISDYGFQIYYSWEDPKLNSIKKIKDLPKRLLIPYKRLDFYIDTILVAKVIKNLIELYRKSYVYETFGEEYGYAKKAEDILFDWDSINLSEGIEQKIQKIKDEFSDLLYEARESKRQNFVESFENILGLYDKNFASDRNSYQEKIQSMIIKKQQENIEQKLKREFKEVIERGFEGMDQNKKYYKVLSPNIKGGLLYTDTNNLGFFRSHLAFMLLSKISDDLYRKNNLVSKGGNKGILDQTPETMLTLEFGKSNLPNISIKRKFFNIKYNSSWIGIRKPKFSIKGAVIREITKKVRDEQRLIKSLEGVWHKSTHFKRWGKPRFNLPRHPDREKNNDDNLMESITSRREQIQLLLREKQKQQEKMAGRLDKIDKEIQNLQTANFQIKQIDKKPALTEKSEGKQSVRNALSAWKWFMEDLIKYQKRTPILQLKLAK M SEQKFSKRQEGFVIPENIGLYKCLAIVRSANLDADVQGHVSCYGVKKNGTYVLKQNGKKSI IDREKGRKYASDLVAFKGDIEKIPFEVIEEKKKEQSIVLGKFNYKLVLDVMKGEKDRASLT NO:MKNKSKKLVQVSSLGTDEFLLTLLNEKFGIEEIYGIIEPEVFSGKKLVCKACQQSTYAPL 76VEYMPVGELDSKYKILSAIKGYDFLSLAYNLARHRSNKKRGHQKLGGGELSEVVISANNAKALNVIKRSLNHYYSEIKPEISKLRKKMQNEPLKVGKQARMRRELHQLSRKVKRLKWKWGKIPNLELQNITFKESDRDFISYALLHTLGRDIGMFNKTEIKMPSNILGYGFQIYYDWEEPKLNTIKKSKNTPKRILIPYKKLDFYNDSILVARAIKELVGLFQESYEWEIFGNEYNYAKEAEVELIKLDEESINGNVEKKLQRIKENFSNLLEKAREKKRQNFIESFESIARLYDESFTADRNEYQREIQSFIIEKQKQSIEKKLKNEFKKIVEKKFNEQEQGKKHYRVLNPTIINEFLPKDKNNLGFLRSKIAFILLSKISDDLYKKSNAVSKGGEKGIIKQQPETILDLEFSKSKLPSINIKKKLFNIKYTSSWLGIRKPKFNIKGAKIREITRRVRDVQRTLKSAESSWYASTHFRRWGFPRFNQPRHPDKEKKSDDRLIESITLLREQIQILLREKQKGQKEMAGRLDDVDKKIQNLQTANFQIKQTGDKPALTEKSAGKQSFRNALSAWKWFMENLLKYQNKTPDLKLKIA RTVM SEQKWIEPNNIDFNKCLAITRSANLDADVQGHKMCYGIKTNGTYKAIGKINKKHNTGIIEKR IDRTYVYDLIVTKEKNEKIVKKTDFMAIDEEIEFDEKKEKLLKKYIKAEVLGTGELIRKDLN NO:DGEKFDDLCSIEEPQAFRRSELVCKACNQSTYASDIRYIPIGEIEAKYKILKAIKGYDFLSL 77KYNLGRLRDSKKRGHQKMGQGELKEFVICANKEKALDVIKRSLNHYLNEVKDEISRLNKKMQNEPLKVNDQARWRRELNQISRRLKRLKWKWGEIPNPELKNLIFKSSRPEFVSYALIHTLGRDIGLINETELKPNNIQEYGFQIYYKWEDPELNHIKKVKNIPKRFIIPYKNLDLFGKYTILSRAIEGILKLYSSSFQYKSFKDPNLFAKEGEKKITNEDFELGYDEKIKKIKDDFKSYKKALLEKKKNTLEDSLNSILSVYEQSLLTEQINNVKKWKEGLLKSKESIHKQKKIENIEDIISRIEELKNVEGWIRTKERDIVNKEETNLKREIKKELKDSYYEEVRKDFSDLKKGEESEKKPFREEPKPIVIKDYIKFDVLPGENSALGFFLSHLSFNLFDSIQYELFEKSRLSSSKFIPQIP ETILDLSEQ FRKFVKRSGAPQPDNLNKYKCIAIVRAANLDADIMSNESSNCVMCKGIKMNKRKTAKG IDAAKTTELGRVYAGQSGNLLCTACTKSTMGPLVDYVPIGRIRAKYTILRAVKEYDFLSLA NO:YNLARTRVSKKGGRQKMHSLSELVIAAEYEIAWNIIKSSVIHYHQETKEEISGLRKKLQA 78EHIHKNKEARIRREMHQISRRIKRLKWKWHMIPNSELHNFLFKQQDPSFVAVALLHTLGRDIGMINKPKGSAKREFIPEYGFQIYYKWMNPKLNDINKQKYRKMPKRSLIPYKNLNVFGDRELIENAMHKLLKLYDENLEVKGSKFFKTRVVAISSKESEKLKRDLLWKGELAKIKKDFNADKNKMQELFKEVKEPKKANALMKQSRNMGFLLQNISYGALGLLANRMYEASAKQSKGDATKQPSIVIPLEMEFGNAFPKLLLRSGKFAMNVSSPWLTIRKPKFVIKGNKIKNITKLMKDEKAKLKRLETSYHRATHFRPTLRGSIDWDSPYFSSPKQPNTHRRSPDRLSADITEYRGRLKSVEAELREGQRAMAKKLDSVDMTASNLQTSNFQLEKGEDPRLTEIDEKGRSIRNCISSWKKFMEDLMKAQEANPVIKIKIALKDESSVLSEDSM SEQKFHPENLNKSYCLAIVRAANLDADIQGHINCIGIKSNKSDRNYENKLESLQNVELLCKA IDCTKSTYKPNINSVPVGEKKAKYSILSEIKKYDFNSLVYNLKKYRKGKSRGHQKLNELRE NO:LVITSEYKKALDVINKSVNHYLVNIKNKMSKLKKILQNEHIHVGTLARIRRERNRISRKL 79DHYRKKWKFVPNKILKNYVFKNQSPDFVSVALLHKLGRDIGLITKTAILQKSFPEYSLQLYYKYDTPKLNYLKKSKFKSLPKRILISYKYPKFDINSNYIEESIDKLLKLYEESPIYKNNSKIIEFFKKSEDNLIKSENDSLKRGIMKEFEKVTKNFSSKKKKLKEELKLKNEDKNSKMLAKVSRPIGFLKAYLSYMLFNIISNRIFEFSRKSSGRIPQLPSCIINLGNQFENFKNELQDSNIGSKKNYKYFCNLLLKSSGFNISYEEEHLSIKTPNFFINGRKLKEITSEKKKIRKENEQLIKQWKKLTFFKPSNLNGKKTSDKIRFKSPNNPDIERKSEDNIVENIAKVKYKLEDLLSEQRKEFNKLAKKHDGVDVEAQCLQTKSFWIDSNSPIKKSLEKKNEKVSVKKKMKAIRSCISAWKWFMADLIEAQKETPMIKLKLALM SEQTTLVPSHLAGIEVMDETTSRNEDMIQKETSRSNEDENYLGVKNKCGINVHKSGRGSSKH IDEPNMPPEKSGEGQMPKQDSTEMQQRFDESVTGETQVSAGATASIKTDARANSGPRVGT NO:ARALIVKASNLDRDIKLGCKPCEYIRSELPMGKKNGCNHCEKSSDIASVPKVESGFRKA 80KYELVRRFESFAADSISRHLGKEQARTRGKRGKKDKKEQMGKVNLDEIAILKNESLIEYTENQILDARSNRIKEWLRSLRLRLRTRNKGLKKSKSIRRQLITLRRDYRKWIKPNPYRPDEDPNENSLRLHTKLGVDIGVQGGDNKRMNSDDYETSFSITWRDTATRKICFTKPKGLLPRHMKFKLRGYPELILYNEELRIQDSQKFPLVDWERIPIFKLRGVSLGKKKVKALNRITEAPRLVVAKRIQVNIESKKKKVLTRYVYNDKSINGRLVKAEDSNKDPLLEFKKQAEEINSDAKYYENQEIAKNYLWGCEGLHKNLLEEQTKNPYLAFKYGFLNIV SEQLDFKRTCSQELVLLPEIEGLKLSGTQGVTSLAKKLINKAANVDRDESYGCHHCIHTRTSL IDSKPVKKDCNSCNQSTNHPAVPITLKGYKIAFYELWHRFTSWAVDSISKALHRNKVMGK NO:VNLDEYAVVDNSHIVCYAVRKCYEKRQRSVRLHKRAYRCRAKHYNKSQPKVGRIYKK 81SKRRNARNLKKEAKRYFQPNEITNGSSDALFYKIGVDLGIAKGTPETEVKVDVSICFQVYYGDARRVLRVRKMDELQSFHLDYTGKLKLKGIGNKDTFTIAKRNESLKWGSTKYEVSRAHKKFKPFGKKGSVKRKCNDYFRSIASWSCEAASQRAQSNLKNAFPYQKALVKCYKNLDYKGVKKNDMWYRLCSNRIFRYSRIAEDIAQYQSDKGKAKFEFVILAQSVAEYDISA IM SEQVFLTDDKRKTALRKIRSAFRKTAEIALVRAQEADSLDRQAKKLTIETVSFGAPGAKNAFI IDGSLQGYNWNSHRANVPSSGSAKDVFRITELGLGIPQSAHEASIGKSFELVGNVVRYTAN NO:LLSKGYKKGAVNKGAKQQREIKGKEQLSFDLISNGPISGDKLINGQKDALAWWLIDKM 82GFHIGLAMEPLSSPNTYGITLQAFWKRHTAPRRYSRGVIRQWQLPFGRQLAPLIHNFFRKKGASIPIVLTNASKKLAGKGVLLEQTALVDPKKWWQVKEQVTGPLSNIWERSVPLVLYTATFTHKHGAAHKRPLTLKVIRISSGSVFLLPLSKVTPGKLVRAWMPDINILRDGRPDEAAYKGPDLIRARERSFPLAYTCVTQIADEWQKRALESNRDSITPLEAKLVTGSDLLQIHSTVQQAVEQGIGGRISSPIQELLAKDALQLVLQQLFMTVDLLRIQWQLKQEVADGNTSEKAVGWAIRISNIHKDAYKTAIEPCTSALKQAWNPLSGFEERTFQLDASIVRKRSTAKTPDDELVIVLRQQAAEMTVAVTQSVSKELMELAVRHSATLHLLVGEVASKQLSRSADKDRG AMDHWKLLSQSMSEQ EDLLQKALNTATNVAAIERHSCISCLFTESEIDVKYKTPDKIGQNTAGCQSCTFRVGYSG IDNSHTLPMGNRIALDKLRETIQRYAWHSLLFNVPPAPTSKRVRAISELRVAAGRERLFTVI NO:TFVQTNILSKLQKRYAANWTPKSQERLSRLREEGQHILSLLESGSWQQKEVVREDQDLI 83VCSALTKPGLSIGAFCRPKYLKPAKHALVLRLIFVEQWPGQIWGQSKRTRRMRRRKDVERVYDISVQAWALKGKETRISECIDTMRRHQQAYIGVLPFLILSGSTVRGKGDCPILKEITRMRYCPNNEGLIPLGIFYRGSANKLLRVVKGSSFTLPMWQNIETLPHPEPFSPEGWTATGALYEKNLAYWSALNEAVDWYTGQILSSGLQYPNQNEFLARLQNVIDSIPRKWFRPQGLKNLKPNGQEDIVPNEFVIPQNAIRAHHVIEWYHKTNDLVAKTLLGWGSQTTLNQTRPQGDLRFTYTRYYFREKEVPEV SEQVPKKKLMRELAKKAVFEAIFNDPIPGSFGCKRCTLIDGARVTDAIEKKQGAKRCAGCEP IDCTFHTLYDSVKHALPAATGCDRTAIDTGLWEILTALRSYNWMSFRRNAVSDASQKQV NO:WSIEELAIWADKERALRVILSALTHTIGKLKNGFSRDGVWKGGKQLYENLAQKDLAKG 84LFANGEIFGKELVEADHDMLAWTIVPNHQFHIGLIRGNWKPAAVEASTAFDARWLTNGAPLRDTRTHGHRGRRFNRTEKLTVLCIKRDGGVSEEFRQERDYELSVMLLQPKNKLKPEPKGELNSFEDLHDHWWFLKGDEATALVGLTSDPTVGDFIQLGLYIRNPIKAHGETKRRLLICFEPPIKLPLRRAFPSEAFKTWEPTINVFRNGRRDTEAYYDIDRARVFEFPETRVSLEHLSKQWEVLRLEPDRENTDPYEAQQNEGAELQVYSLLQEAAQKMAPKVVIDPFGQFPLELFSTFVAQLFNAPLSDTKAKIGKPLDSGFVVESHLHLLEEDFAYRDFVRVTFMGTEPTFRVIHYSNGEGYWKKTVLKGKNNIRTALIPEGAKAAVDAYKNKRCPLTLEAAILNEEKDRRLVLGNKALSLLAQTARGNLTILEALAAEVLRPLSGTEGVVHLHACVTRHSTLTESTET DNM SEQVEKLFSERLKRAMWLKNEAGRAPPAETLTLKHKRVSGGHEKVKEELQRVLRSLSGTNQ IDAAWNLGLSGGREPKSSDALKGEKSRVVLETVVFHSGHNRVLYDVIEREDQVHQRSSIM NO:HMRRKGSNLLRLWGRSGKVRRKMREEVAEIKPVWHKDSRWLAIVEEGRQSVVGISSA 85GLAVFAVQESQCTTAEPKPLEYVVSIWFRGSKALNPQDRYLEFKKLKTTEALRGQQYDPIPFSLKRGAGCSLAIRGEGIKFGSRGPIKQFFGSDRSRPSHADYDGKRRLSLFSKYAGDLADLTEEQWNRTVSAFAEDEVRRATLANIQDFLSISHEKYAERLKKRIESIEEPVSASKLEAYLSAIFETFVQQREALASNFLMRLVESVALLISLEEKSPRVEFRVARYLAESKEGFNRK AM SEQVVITQSELYKERLLRVMEIKNDRGRKEPRESQGLVLRFTQVTGGQEKVKQKLWLIFEGF IDSGTNQASWNFGQPAGGRKPNSGDALKGPKSRVTYETVVFHFGLRLLSAVIERHNLKQQ NO:RQTMAYMKRRAAARKKWARSGKKCSRMRNEVEKIKPKWHKDPRWFDIVKEGEPSIV 86GISSAGFAIYIVEEPNFPRQDPLEIEYAISIWFRRDRSQYLTFKKIQKAEKLKELQYNPIPFRLKQEKTSLVFESGDIKFGSRGSIEHFRDEARGKPPKADMDNNRRLTMFSVFSGNLTNLTEEQYARPVSGLLAPDEKRMPTLLKKLQDFFTPIHEKYGERIKQRLANSEASKRPFKKLEEYLPAIYLEFRARREGLASNWVLVLINSVRTLVRIKSEDPYIEFKVSQYLLEKEDNKAL SEQKQDALFEERLKKAIFIKRQADPLQREELSLLPPNRKIVTGGHESAKDTLKQILRAINGTN IDQASWNPGTPSGKRDSKSADALAGPKSRVKLETVVFHVGHRLLKKVVEYQGHQKQQH NO:GLKAFMRTCAAMRKKWKRSGKVVGELREQLANIQPKWHYDSRPLNLCFEGKPSVVGL 87RSAGIALYTIQKSVVPVKEPKPIEYAVSIWFRGPKAMDREDRCLEFKKLKIATELRKLQFEPIVSTLTQGIKGFSLYIQGNSVKFGSRGPIKYFSNESVRQRPPKADPDGNKRLALFSKFSGDLSDLTEEQWNRPILAFEGIIRRATLGNIQDYLTVGHEQFAISLEQLLSEKESVLQMSIEQQRLKKNLGKKAENEWVESFGAEQARKKAQGIREYISGFFQEYCSQREQWAENWVQQLNKSVRLFLTIQDSTPFIEFRVARYLPKGEKKKGKAM SEQANHAERHKRLRKEANRAANRNRPLVADCDTGDPLVGICRLLRRGDKMQPNKTGCRSC IDEQVEPELRDAILVSGPGRLDNYKYELFQRGRAMAVHRLLKRVPKLNRPKKAAGNDEK NO:KAENKKSEIQKEKQKQRRMMPAVSMKQVSVADFKHVIENTVRHLFGDRRDREIAECA 88ALRAASKYFLKSRRVRPRKLPKLANPDHGKELKGLRLREKRAKLKKEKEKQAELARSNQKGAVLHVATLKKDAPPMPYEKTQGRNDYTTFVISAAIKVGATRGTKPLLTPQPREWQCSLYWRDGQRWIRGGLLGLQAGIVLGPKLNRELLEAVLQRPIECRMSGCGNPLQVRGAAVDFFMTTNPFYVSGAAYAQKKFKPFGTKRASEDGAAAKAREKLMTQLAKVLDKVVTQAAHSPLDGIWETRPEAKLRAMIMALEHEWIFLRPGPCHNAAEEVIKCDCTGGHAILWALIDEARGALEHKEFYAVTRAHTHDCEKQKLGGRLAGFLDLLIAQDVPLDDAPAARKIKTLLEATPPAPCYKAATSIATCDCEGKFDKLWAIIDATRAGHGTEDLWARTLAYPQNVNCKCKAGKDLTHRLADFLGLLIKRDGPFRERPPEIKVTGDRKLVFSGDKKCKGHQYVILAKAHNEEVVRAWISRWGLKSRTNKAGYAATELNLLLNWLSICRRRWMDMLTVQRDTPYIRMKTGRLVVDDKKERKAM SEQAKQREALRVALERGIVRASNRTYTLVTNCTKGGPLPEQCRMIERGKARAMKWEPKLV IDGCGSCAAATVDLPAIEEYAQPGRLDVAKYKLTTQILAMATRRMMVRAAKLSRRKGQW NO:PAKVQEEKEEPPEPKKMLKAVEMRPVAIVDFNRVIQTTIEHLWAERANADEAELKALK 89AAAAYFGPSLKIRARGPPKAAIGRELKKAHRKKAYAERKKARRKRAELARSQARGAAAHAAIRERDIPPMAYERTQGRNDVTTIPIAAAIKIAATRGARPLPAPKPMKWQCSLYWNEGQRWIRGGMLTAQAYAHAANIHRPMRCEMWGVGNPLKVRAFEGRVADPDGAKGRKAEFRLQTNAFYVSGAAYRNKKFKPFGTDRGGIGSARKKRERLMAQLAKILDKVVSQAAHSPLDDIWHTRPAQKLRAMIKQLEHEWMFLRPQAPTVEGTKPDVDVAGNMQRQIKALMAPDLPPIEKGSPAKRFTGDKRKKGERAVRVAEAHSDEVVTAWISRWGIQTRRNEGSYAAQELELLLNWLQICRRRWLDMTAAQRVSPYIRMKSGRMITDAADEGVAPIPLVENM SEQKSISGRSIKHMACLKDMLKSEITEIEEKQKKESLRKWDYYSKFSDEILFRRNLNVSANHD IDANACYGCNPCAFLKEVYGFRIERRNNERIISYRRGLAGCKSCVQSTGYPPIEFVRRKFGA NO:DKAMEIVREVLHRRNWGALARNIGREKEADPILGELNELLLVDARPYFGNKSAANETN 90LAFNVITRAAKKFRDEGMYDIHKQLDIHSEEGKVPKGRKSRLIRIERKHKAIHGLDPGETWRYPHCGKGEKYGVWLNRSRLIHIKGNEYRCLTAFGTTGRRMSLDVACSVLGHPLVKKKRKKGKKTVDGTELWQIKKATETLPEDPIDCTFYLYAAKPTKDPFILKVGSLKAPRWKKLHKDFFEYSDTEKTQGQEKGKRVVRRGKVPRILSLRPDAKFKVSIWDDPYNGKNKEGTLLRMELSGLDGAKKPLILKRYGEPNTKPKNFVFWRPHITPHPLTFTPKHDFGDPNKKTKRRRVFNREYYGHLNDLAKMEPNAKFFEDREVSNKKNPKAKNIRIQAKESLPNIVAKNGRWAAFDPNDSLWKLYLHWRGRRKTIKGGISQEFQEFKERLDLYKKHEDESEWKEKEKLWENHEKEWKKTLEIHGSIAEVSQRCVMQSMMGPLDGLVQKKDYVHIGQSSLKAADDAWTFSANRYKKATGPKWGKISVSNLLYDANQANAELISQSISKYLSKQKDNQGCEGRKMKFLIKIIEPLRENFVKHTRWLHEMTQKDCEVRAQFSRVSM SEQFPSDVGADALKHVRMLQPRLTDEVRKVALTRAPSDRPALARFAAVAQDGLAFVRHLN IDVSANHDSNCTFPRDPRDPRRGPCEPNPCAFLREVWGFRIVARGNERALSYRRGLAGCKS NO:CVQSTGFPSVPFHRIGADDCMRKLHEILKARNWRLLARNIGREREADPLLTELSEYLLV 91DARTYPDGAAPNSGRLAENVIKRAAKKFRDEGMRDIHAQLRVHSREGKVPKGRLQRLRRIERKHRAIHALDPGPSWEAEGSARAEVQGVAVYRSQLLRVGEIHTQQIEPVGIVARTLFGVGRTDLDVAVSVLGAPLTKRKKGSKTLESTEDFRIAKARETRAEDKIEVAFVLYPTASLLRDEIPKDAFPAMRIDRFLLKVGSVQADREILLQDDYYRFGDAEVKAGKNKGRTVTRPVKVPRLQALRPDAKFRVNVWADPFGAGDSPGTLLRLEVSGVTRRSQPLRLLRYGQPSTQPANFLCWRPHRVPDPMTFTPRQKFGERRKNRRTRRPRVFERLYQVHIKHLAHLEPNRKWFEEARVSAQKWAKARAIRRKGAEDIPVVAPPAKRRWAALQPNAELWDLYAHDREARKRFRGGRAAEGEEFKPRLNLYLAHEPEAEWESKRDRWERYEKKWTAVLEEHSRMCAVADRTLPQFLSDPLGARMDDKDYAFVGKSALAVAEAFVEEGTVERAQGNCSITAKKKFASNASRKRLSVANLLDVSDKADRALVFQAVRQYVQRQAENGGVEGRRMAFLRKLLAPLRQNFVCHTRWLHM SEQAARKKKRGKIGITVKAKEKSPPAAGPFMARKLVNVAANVDGVEVHLCVECEADAHGS IDASARLLGGCRSCTGSIGAEGRLMGSVDVDRERVIAEPVHTETERLGPDVKAFEAGTAES NO:KYAIQRGLEYWGVDLISRNRARTVRKMEEADRPESSTMEKTSWDEIAIKTYSQAYHAS 92ENHLFWERQRRVRQHALALFRRARERNRGESPLQSTQRPAPLVLAALHAEAAAISGRARAEYVLRGPSANVRAAAADIDAKPLGHYKTPSPKVARGFPVKRDLLRARHRIVGLSRAYFKPSDVVRGTSDAIAHVAGRNIGVAGGKPKEIEKTFTLPFVAYWEDVDRVVHCSSFKADGPWVRDQRIKIRGVSSAVGTFSLYGLDVAWSKPTSFYIRCSDIRKKFHPKGFGPMKHWRQWAKELDRLTEQRASCVVRALQDDEELLQTMERGQRYYDVFSCAATHATRGEADPSGGCSRCELVSCGVAHKVTKKAKGDTGIEAVAVAGCSLCESKLVGPSKPRVHRQMAALRQSHALNYLRRLQREWEALEAVQAPTPYLRFKYARHLEVRSM SEQAAKKKKQRGKIGISVKPKEGSAPPADGPFMARKLVNVAANVDGVEVNLCIECEADAH IDGSAPARLLGGCKSCTGSIGAEGRLMGSVDVDRADAIAKPVNTETEKLGPDVQAFEAGT NO:AETKYALQRGLEYWGVDLISRNRSRTVRRTEEGQPESATMEKTSWDEIAIKSYTRAYH 93ASENHLFWERQRRVRQHALALFKRAKERNRGDSTLPREPGHGLVAIAALACEAYAVGGRNLAETVVRGPTFGTARAVRDVEIASLGRYKTPSPKVAHGSPVKRDFLRARHRIVGLARAYYRPSDVVRGTSDAIAHVAGRNIGVAGGKPRAVEAVFTLPFVAYWEDVDRVVHCSSFQVSAPWNRDQRMKIAGVTTAAGTFSLHGGELKWAKPTSFYIRCSDTRRKFRPKGFGPMKRWRQWAKDLDRLVEQRASCVVRALQDDAALLETMERGQRYYDVFACAVTHATRGEADRLAGCSRCALTPCQEAHRVTTKPRGDAGVEQVQTSDCSLCEGKLVGPSKPRLHRTLTLLRQEHGLNYLRRLQREWESLEAVQVPTPYLRFKYARHLEVRSM SEQTDSQSESVPEVVYALTGGEVPGRVPPDGGSAEGARNAPTGLRKQRGKIKISAKPSKPGS IDPASSLARTLVNEAANVDGVQSSGCATCRMRANGSAPRALPIGCVACASSIGRAPQEETV NO:CALPTTQGPDVRLLEGGHALRKYDIQRALEYWGVDLIGRNLDRQAGRGMEPAEGATA 94TMKRVSMDELAVLDFGKSYYASEQHLFAARQRRVRQHAKALKIRAKHANRSGSVKRALDRSRKQVTALAREFFKPSDVVRGDSDALAHVVGRNLGVSRHPAREIPQTFTLPLCAYWEDVDRVISCSSLLAGEPFARDQEIRIEGVSSALGSLRLYRGAIEWHKPTSLYIRCSDTRRKFRPRGGLKKRWRQWAKDLDRLVEQRACCIVRSLQADVELLQTMERAQRFYDVHDCAATHVGPVAVRCSPCAGKQFDWDRYRLLAALRQEHALNYLRRLQREWESLEAQQVKMPYLRFKYARKLEVSGPLIGLEVRREPSMGTAIAEM SEQAGTAGRRHGSLGARRSINIAGVTDRHGRWGCESCVYTRDQAGNRARCAPCDQSTYAP IDDVQEVTIGQRQAKYTIFLTLQSFSWTNTMRNNKRAAAGRSKRTTGKRIGQLAEIKITGV NO:GLAHAHNVIQRSLQHNITKMWRAEKGKSKRVARLKKAKQLTKRRAYFRRRMSRQSRG 95NGFFRTGKGGIHAVAPVKIGLDVGMIASGSSEPADEQTVTLDAIWKGRKKKIRLIGAKGELAVAACRFREQQTKGDKCIPLILQDGEVRWNQNNWQCHPKKLVPLCGLEVSRKFVSQADRLAQNKVASPLAARFDKTSVKGTLVESDFAAVLVNVTSIYQQCHAMLLRSQEPTPS LRVQRTITSMSEQ GVRFSPAQSQVFFRTVIPQSVEARFAINMAAIHDAAGAFGCSVCRFEDRTPRNAKAVHG IDCSPCTRSTNRPDVFVLPVGAIKAKYDVFMRLLGFNWTHLNRRQAKRVTVRDRIGQLDE NO:LAISMLTGKAKAVLKKSICHNVDKSFKAMRGSLKKLHRKASKTGKSQLRAKLSDLRER 96TNTTQEGSHVEGDSDVALNKIGLDVGLVGKPDYPSEESVEVVVCLYFVGKVLILDAQGRIRDMRAKQYDGFKIPIIQRGQLTVLSVKDLGKWSLVRQDYVLAGDLRFEPKISKDRKYAECVKRIALITLQASLGFKERIPYYVTKQVEIKNASHIAFVTEAIQNCAENFREMTEYLMKYQEKSPDLKVLLTQLM SEQRAVVGKVFLEQARRALNLATNFGTNHRTGCNGCYVTPGKLSIPQDGEKNAAGCTSCL IDMKATASYVSYPKPLGEKVAKYSTLDALKGFPWYSLRLNLRPNYRGKPINGVQEVAPVS NO:KFRLAEEVIQAVQRYHFTELEQSFPGGRRRLRELRAFYTKEYRRAPEQRQHVVNGDRNI 97VVVTVLHELGFSVGMFNEVELLPKTPIECAVNVFIRGNRVLLEVRKPQFDKERLLVESLWKKDSRRHTAKWTPPNNEGRIFTAEGWKDFQLPLLLGSTSRSLRAIEKEGFVQLAPGRDPDYNNTIDEQHSGRPFLPLYLYLQGTISQEYCVFAGTWVIPFQDGISPYSTKDTFQPDLKRKAYSLLLDAVKHRLGNKVASGLQYGRFPAIEELKRLVRMHGATRKIPRGEKDLLKKGDPDTPEWWLLEQYPEFWRLCDAAAKRVSQNVGLLLSLKKQPLWQRRWLESRTRNEPLDNLPLSMALTLHLTNEEAL SEQAAVYSKFYIENHFKMGIPETLSRIRGPSIIQGFSVNENYINIAGVGDRDFIFGCKKCKYTR IDGKPSSKKINKCHPCKRSTYPEPVIDVRGSISEFKYKIYNKLKQEPNQSIKQNTKGRMNPS NO:DHTSSNDGIIINGIDNRIAYNVIFSSYKHLMEKQINLLRDTTKRKARQIKKYNNSGKKKH 98SLRSQTKGNLKNRYHMLGMFKKGSLTITNEGDFITAVRKVGLDISLYKNESLNKQEVETELCLNIKWGRTKSYTVSGYIPLPINIDWKLYLFEKETGLTLRLFGNKYKIQSKKFLIAQLFKPKRPPCADPVVKKAQKWSALNAHVQQMAGLFSDSHLLKRELKNRMHKQLDFKSLWVGTEDYIKWFEELSRSYVEGAEKSLEFFRQDYFCFNYTKQTTM SEQPQQQRDLMLMAANYDQDYGNGCGPCTVVASAAYRPDPQAQHGCKRHLRTLGASAVT IDHVGLGDRTATITALHRLRGPAALAARARAAQAASAPMTPDTDAPDDRRRLEAIDADDV NO:VLVGAHRALWSAVRRWADDRRAALRRRLHSEREWLLKDQIRWAELYTLIEASGTPPQ 99GRWRNTLGALRGQSRWRRVLAPTMRATCAETHAELWDALAELVPEMAKDRRGLLRPPVEADALWRAPMIVEGWRGGHSVVVDAVAPPLDLPQPCAWTAVRLSGDPRQRWGLHLAVPPLGQVQPPDPLKATLAVSMRHRGGVRVRTLQAMAVDADAPMQRHLQVPLTLQRGGGLQWGIHSRGVRRREARSMASWEGPPIWTGLQLVNRWKGQGSALLAPDRPPDTPPYAPDAAVAPAQPDTKRARRTLKEACTVCRCAPGHMRQLQVTLTGDGTWRRFRLRAPQGAKRKAEVLKVATQHDERIANYTAWYLKRPEHAAGCDTCDGDSRLDGACRGCRPLLVGDQCFRRYLDKIEADRDDGLAQIKPKAQEAVAAMAAKRDARAQKVAARAAKLSEATGQRTAATRDASHEARAQKELEAVATEGTTVRHDAAAVSAFGSWVARKGDEYRHQVGVLANRLEHGLRLQELMAPDSVVADQQRASGHARVGYRYVLTAM SEQAVAHPVGRGNAGSPGARGPEELPRQLVNRASNVTRPATYGCAPCRHVRLSIPKPVLTG IDCRACEQTTHPAPKRAVRGGADAAKYDLAAFFAGWAADLEGRNRRRQVHAPLDPQPDP NO:NHEPAVTLQKIDLAEVSIEEFQRVLARSVKHRHDGRASREREKARAYAQVAKKRRNSH 100AHGARTRRAVRRQTRAVRRAHRMGANSGEILVASGAEDPVPEAIDHAAQLRRRIRACARDLEGLRHLSRRYLKTLEKPCRRPRAPDLGRARCHALVESLQAAERELEELRRCDSPDTAMRRLDAVLAAAASTDATFATGWTVVGMDLGVAPRGSAAPEVSPMEMAISVFWRKGSRRVIVSKPIAGMPIRRHELIRLEGLGTLRLDGNHYTGAGVTKGRGLSEGTEPDFREKSPSTLGFTLSDYRHESRWRPYGAKQGKTARQFFAAMSRELRALVEHQVLAPMGPPLLEAHERRFETLLKGQDNKSIHAGGGGRYVWRGPPDSKKRPAADGDWFRFGRGHADHRGWANKRHELAANYLQSAFRLWSTLAEAQEPTPYARYKYTRVTM SEQWDFLTLQVYERHTSPEVCVAGNSTKCASGTRKSDHTHGVGVKLGAQEINVSANDDRD IDHEVGCNICVISRVSLDIKGWRYGCESCVQSTPEWRSIVRFDRNHKEAKGECLSRFEYWG NO:AQSTARSLKRNKLMGGVNLDELAIVQNENVVKTSLKHLFDKRKDRIQANLKAVKVRM 101RERRKSGRQRKALRRQCRKLKRYLRSYDPSDIKEGNSCSAFTKLGLDIGISPNKPPKIEPKVEVVFSLFYQGACDKIVTVSSPESPLPRSWKIKIDGIRALYVKSTKVKFGGRTFRAGQRNNRRKVRPPNVKKGKRKGSRSQFFNKFAVGLDAVSQQLPIASVQGLWGRAETKKAQTICLKQLESNKPLKESQRCLFLADNWVVRVCGFLRALSQRQGPTPYIRYRYRCNM SEQARNVGQRNASRQSKRESAKARSRRVTGGHASVTQGVALINAAANADRDHTTGCEPCT IDWERVNLPLQEVIHGCDSCTKSSPFWRDIKVVNKGYREAKEEIMRIASGISADHLSRALS NO:HNKVMGRLNLDEVCILDFRTVLDTSLKHLTDSRSNGIKEHIRAVHRKIRMRRKSGKTAR 102ALRKQYFALRRQWKAGHKPNSIREGNSLTALRAVGFDVGVSEGTEPMPAPQTEVVLSVFYKGSATRILRISSPHPIAKRSWKVKIAGIKALKLIRREHDFSFGRETYNASQRAEKRKFSPHAARKDFFNSFAVQLDRLAQQLCVSSVENLWVTEPQQKLLTLAKDTAPYGIREGARFADTRARLAWNWVFRVCGFTRALHQEQEPTPYCRFTWRSKM

In some embodiments, the Type VI CRISPR/Cas enzyme is a programmableCas13 nuclease. The general architecture of a Cas13 protein includes anN-terminal domain and two HEPN (higher eukaryotes and prokaryotesnucleotide-binding) domains separated by two helical domains (Liu etal., Cell 2017 Jan. 12; 168(1-2):121-134.el2). The HEPN domains eachcomprise aR-X₄-H motif. Shared features across Cas13 proteins includethat upon binding of the crRNA of the guide nucleic acid to a targetnucleic acid, the protein undergoes a conformational change to bringtogether the HEPN domains and form a catalytically active RNase. (Tambeet al., Cell Rep. 2018 Jul. 24; 24(4): 1025-1036). Thus, two activatableHEPN domains are characteristic of a programmable Cas13 nuclease of thepresent disclosure. In some embodiments, a programmable nuclease (e.g.,a Cas13 programmable nuclease) comprises at least two HEPN domains.However, programmable Cas13 nucleases also consistent with the presentdisclosure include Cas13 nucleases comprising mutations in the HEPNdomain that enhance the Cas13 proteins cleavage efficiency or mutationsthat catalytically inactivate the HEPN domains. Programmable Cas13nucleases consistent with the present disclosure also Cas13 nucleasecomprising catalytic

A programmable Cas13 nuclease can be a Cas13a protein (also referred toas “c2c2”), a Cas13b protein, a Cas13c protein, a Cas13d protein, or aCas13e protein. Example C2c2 proteins are set forth as SEQ ID NO:103-SEQ ID NO: 110. Example Cas13b proteins are set forth in SEQ ID NO:128-SEQ ID NO: 132. Example Cas13c proteins are set forth in SEQ ID NO:133-SEQ ID NO: 137. In some cases, a subject C2c2 protein includes anamino acid sequence having 80% or more (e.g., 85% or more, 90% or more,95% or more, 98% or more, 99% or more, 99.5% or more, or 100%) aminoacid sequence identity with the amino acid sequence set forth in any oneof SEQ ID NO: 103-SEQ ID NO: 110. In some embodiments, a programmablenuclease has at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 92%, at least 95%, at least 97%, at least 99%, or100% sequence identity to any one of SEQ ID NO: 103-SEQ ID NO: 137. Insome embodiments, the programmable nuclease has at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 92%, at least95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:104. In some cases, a suitable C2c2 polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to theListeria seeligeri C2c2 amino acid sequence set forth in SEQ ID NO: 103.In some cases, a suitable C2c2 polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to theLeptotrichia buccalis C2c2 amino acid sequence set forth in SEQ ID NO:104. In some cases, a suitable C2c2 polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to theRhodobacter capsulatus C2c2 amino acid sequence set forth in SEQ ID NO:106. In some cases, a suitable C2c2 polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to theCarnobacterium gallinarum C2c2 amino acid sequence set forth in SEQ IDNO: 107. In some cases, a suitable C2c2 polypeptide comprises an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the Herbinix hemicellulosilytica C2c2 amino acid sequence set forthin SEQ ID NO: 108. In some cases, the C2c2 protein includes an aminoacid sequence having 80% or more amino acid sequence identity with theLeptotrichia buccalis (Lbu) C2c2 amino acid sequence set forth in SEQ IDNO: 104. In some cases, the C2c2 protein is a Leptotrichia buccalis(Lbu) C2c2 protein (e.g., see SEQ ID NO: 104). In some cases, the C2c2protein includes the amino acid sequence set forth in any one of SEQ IDNOs: 103-104 and SEQ ID NOs: 106-110. In some cases, a C2c2 protein usedin a method of the present disclosure is not a Leptotrichia shahii (Lsh)C2c2 protein. In some cases, a C2c2 protein used in a method of thepresent disclosure is not a C2c2 polypeptide having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the Lsh C2c2 polypeptide set forthin SEQ ID NO: 105.

TABLE 3 Cas13 Sequences SEQ ID NO Description Sequence SEQ ListeriaMWISIKTLIHHLGVLFFCDYMYNRREKKIIEVKTMRITKVEVDRKKV ID seeligeri C2c2LISRDKNGGKLVYENEMQDNTEQIMHHKKSSFYKSVVNKTICRPEQ NO: amino acidKQMKKLVHGLLQENSQEKIKVSDVTKLNISNFLNHRFKKSLYYFPE 103 sequenceNSPDKSEEYRIEINLSQLLEDSLKKQQGTFICWESFSKDMELYINWAENYISSKTKLIKKSIRNNRIQSTESRSGQLMDRYMKDILNKNKPFDIQSVSEKYQLEKLTSALKATFKEAKKNDKEINYKLKSTLQNHERQIIEELKENSELNQFNIEIRKHLETYFPIKKTNRKVGDIRNLEIGEIQKIVNHRLKNKIVQRILQEGKLASYEIESTVNSNSLQKIKIEEAFALKFINACLFASNNLRNMVYPVCKKDILMIGEFKNSFKEIKHKKFIRQWSQFFSQEITVDDIELASWGLRGAIAPIRNEIIHLKKHSWKKFFNNPTFKVKKSKIINGKTKDVTSEFLYKETLFKDYFYSELDSVPELIINKMESSKILDYYSSDQLNQVFTIPNFELSLLTSAVPFAPSFKRVYLKGFDYQNQDEAQPDYNLKLNIYNEKAFNSEAFQAQYSLFKMVYYQVFLPQFTTNNDLFKSSVDFILTLNKERKGYAKAFQDIRKMNKDEKPSEYMSYIQSQLMLYQKKQEEKEKINHFEKFINQVFIKGFNSFIEKNRLTYICHPTKNTVPENDNIEIPFHTDMDDSNIAFWLMCKLLDAKQLSELRNEMIKFSCSLQSTEEISTFTKAREVIGLALLNGEKGCNDWKELFDDKEAWKKNMSLYVSEELLQSLPYTQEDGQTPVINRSIDLVKKYGTETILEKLFSSSDDYKVSAKDIAKLHEYDVTEKIAQQESLHKQWIEKPGLARDSAWTKKYQNVINDISNYQWAKTKVELTQVRHLHQLTIDLLSRLAGYMSIADRDFQFSSNYILERENSEYRVTSWILLSENKNKNKYNDYELYNLKNASIKVSSKNDPQLKVDLKQLRLTLEYLELFDNRLKEKRNNISHFNYLNGQLGNSILELFDDARDVLSYDRKLKNAVSKSLKEILSSHGMEVTFKPLYQTNHHLKIDKLQPKKIHHLGEKSTVSSNQVSNEYCQLVRTLLTMK SEQ LeptotrichiaMKVTKVGGISHKKYTSEGRLVKSESEENRTDERLSALLNMRLDMYI ID buccalis (Lbu)KNPSSTETKENQKRIGKLKKFFSNKMVYLKDNTLSLKNGKKENIDR NO: C2c2 aminoEYSETDILESDVRDKKNFAVLKKIYLNENVNSEELEVFRNDIKKKLN 104 acid sequenceKINSLKYSFEKNKANYQKINENNIEKVEGKSKRNIIYDYYRESAKRDAYVSNVKEAFDKLYKEEDIAKLVLEIENLTKLEKYKIREFYHEIIGRKNDKENFAKIIYEEIQNVNNMKELIEKVPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQDGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKKNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKIFRQLNSANVFRYLEKYKILNYLKRTRFEFVNKNIPFVPSFTKLYSRIDDLKNSLGIYWKTPKTNDDNKTKEIIDAQIYLLKNIYYGEFLNYFMSNNGNFFEISKEIIELNKNDKRNLKTGFYKLQKFEDIQEKIPKEYLANIQSLYMINAGNQDEEEKDTYIDFIQKIFLKGFMTYLANNGRLSLIYIGSDEETNTSLAEKKQEFDKFLKKYEQNNNIKIPYEINEFLREIKLGNILKYTERLNMFYLILKLLNHKELTNLKGSLEKYQSANKEEAFSDQLELINLLNLDNNRVTEDFELEADEIGKFLDFNGNKVKDNKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAGYKISIEELKKYSNKKNEIEKNHKMQENLHRKYARPRKDEKFTDEDYESYKQAIENIEEYTHLKNKVEFNELNLLQGLLLRILHRLVGYTSIWERDLRFRLKGEFPENQYIEEIFNFENKKNVKYKGGQIVEKYIKFYKELHQNDEVKINKYSSANIKVLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAVMKSVVDILKEYGFVATFKIGADKKIGIQTLESEKIVHLKNLKKKKLMTDRNSEELCKLVKIMFEYKMEEKKSEN SEQ LeptotrichiaMGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENN ID shahii (Lsh)NKEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIR NO: C2c2 proteinIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKD 105DKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIFNNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDT L SEQ RhodobacterMQIGKVQGRTISEFGDPAGGLKRKISTDGKNRKELPAHLSSDPKALI ID capsulatusGQWISGIDKIYRKPDSRKSDGKAIHSPTPSKMQFDARDDLGEAFWK NO: C2c2 aminoLVSEAGLAQDSDYDQFKRRLHPYGDKFQPADSGAKLKFEADPPEPQ 106 acid sequenceAFHGRWYGAMSKRGNDAKELAAALYEHLHVDEKRIDGQPKRNPKTDKFAPGLVVARALGIESSVLPRGMARLARNWGEEEIQTYFVVDVAASVKEVAKAAVSAAQAFDPPRQVSGRSLSPKVGFALAEHLERVTGSKRCSFDPAAGPSVLALHDEVKKTYKRLCARGKNAARAFPADKTELLALMRHTHENRVRNQMVRMGRVSEYRGQQAGDLAQSHYWTSAGQTEIKESEIFVRLWVGAFALAGRSMKAWIDPMGKIVNTEKNDRDLTAAVNIRQVISNKEMVAEAMARRGIYFGETPELDRLGAEGNEGFVFALLRYLRGCRNQTFHLGARAGFLKEIRKELEKTRWGKAKEAEHVVLTDKTVAAIRAIIDNDAKALGARLLADLSGAFVAHYASKEHFSTLYSEIVKAVKDAPEVSSGLPRLKLLLKRADGVRGYVHGLRDTRKHAFATKLPPPPAPRELDDPATKARYIALLRLYDGPFRAYASGITGTALAGPAARAKEAATALAQSVNVTKAYSDVMEGRSSRLRPPNDGETLREYLSALTGETATEFRVQIGYESDSENARKQAEFIENYRRDMLAFMFEDYIRAKGFDWILKIEPGATAMTRAPVLPEPIDTRGQYEHWQAALYLVMHFVPASDVSNLLHQLRKWEALQGKYELVQDGDATDQADARREALDLVKRFRDVLVLFLKTGEARFEGRAAPFDLKPFRALFANPATFDRLFMATPTTARPAEDDPEGDGASEPELRVARTLRGLRQIARYNHMAVLSDLFAKHKVRDEEVARLAEIEDETQEKSQIVAAQELRTDLHDKVMKCHPKTISPEERQSYAAAIKTIEEHRFLVGRVYLGDHLRLHRLMMDVIGRLIDYAGAYERDTGTFLINASKQLGAGADWAVTIAGAANTDARTQTRKDLAHFNVLDRADGTPDLTALVNRAREMMAYDRKRKNAVPRSILDMLARLGLTLKWQMKDHLLQDATITQAAIKHLDKVRLTVGGPAAVTEARFSQDYLQMVAAVFNGSVQNPKPRRRDDGDAWHKPPKPATAQSQPDQKPPNKAPSAGSRLPPPQVGEVYEGVVVKVIDTGSLGFLAVEGVAGNIGLHISRLRRIREDAIIVGRRYRFRVEIYVPPKSNTSKL NAADLVRID SEQCarnobacterium MRITKVKIKLDNKLYQVTMQKEEKYGTLKLNEESRKSTAEILRLKK IDgallinarum ASFNKSFHSKTINSQKENKNATIKKNGDYISQIFEKLVGVDTNKNIR NO:C2c2 amino KPKMSLTDLKDLPKKDLALFIKRKFKNDDIVEIKNLDLISLFYNALQ 107acid sequence KVPGEHFTDESWADFCQEMMPYREYKNKFIERKIILLANSIEQNKGFSINPETFSKRKRVLHQWAIEVQERGDFSILDEKLSKLAEIYNFKKMCKRVQDELNDLEKSMKKGKNPEKEKEAYKKQKNFKIKTIWKDYPYKTHIGLIEKIKENEELNQFNIEIGKYFEHYFPIKKERCTEDEPYYLNSETIATTVNYQLKNALISYLMQIGKYKQFGLENQVLDSKKLQEIGIYEGFQTKFMDACVFATSSLKNIIEPMRSGDILGKREFKEAIATSSFVNYHHFFPYFPFELKGMKDRESELIPFGEQTEAKQMQNIWALRGSVQQIRNEIFHSFDKNQKFNLPQLDKSNFEFDASENSTGKSQSYIETDYKFLFEAEKNQLEQFFIERIKSSGALEYYPLKSLEKLFAKKEMKFSLGSQVVAFAPSYKKLVKKGHSYQTATEGTANYLGLSYYNRYELKEESFQAQYYLLKLIYQYVFLPNFSQGNSPAFRETVKAILRINKDEARKKMKKNKKFLRKYAFEQVREMEFKETPDQYMSYLQSEMREEKVRKAEKNDKGFEKNITMNFEKLLMQIFVKGFDVFLTTFAGKELLLSSEEKVIKETEISLSKKINEREKTLKASIQVEHQLVATNSAISYWLFCKLLDSRHLNELRNEMIKFKQSRIKFNHTQHAELIQNLLPIVELTILSNDYDEKNDSQNVDVSAYFEDKSLYETAPYVQTDDRTRVSFRPILKLEKYHTKSLIEALLKDNPQFRVAATDIQEWMHKREEIGELVEKRKNLHTEWAEGQQTLGAEKREEYRDYCKKIDRFNWKANKVTLTYLSQLHYLITDLLGRMVGFSALFERDLVYFSRSFSELGGETYHISDYKNLSGVLRLNAEVKPIKIKNIKVIDNEENPYKGNEPEVKPFLDRLHAYLENVIGIKAVHGKIRNQTAHLSVLQLELSMIESMNNLRDLMAYDRKLKNAVTKSMIKILDKHGMILKLKIDENHKNFEIESLIPKEIIHLKDKAIKTNQVSEEYCQLVLALLTTNPG NQLN SEQ HerbinixMKLTRRRISGNSVDQKITAAFYRDMSQGLLYYDSEDNDCTDKVIES ID hemicellulosilyticaMDFERSWRGRILKNGEDDKNPFYMFVKGLVGSNDKIVCEPIDVDSD NO: C2c2PDNLDILINKNLTGFGRNLKAPDSNDTLENLIRKIQAGIPEEEVLPEL 108 amino acidKKIKEMIQKDIVNRKEQLLKSIKNNRIPFSLEGSKLVPSTKKMKWLF sequenceKLIDVPNKTFNEKMLEKYWEIYDYDKLKANITNRLDKTDKKARSISRAVSEELREYHKNLRTNYNRFVSGDRPAAGLDNGGSAKYNPDKEEFLLFLKEVEQYFKKYFPVKSKHSNKSKDKSLVDKYKNYCSYKVVKKEVNRSIINQLVAGLIQQGKLLYYFYYNDTWQEDFLNSYGLSYIQVEEAFKKSVMTSLSWGINRLTSFFIDDSNTVKFDDITTKKAKEAIESNYFNKLRTCSRMQDHFKEKLAFFYPVYVKDKKDRPDDDIENLIVLVKNAIESVSYLRNRTFHFKESSLLELLKELDDKNSGQNKIDYSVAAEFIKRDIENLYDVFREQIRSLGIAEYYKADMISDCFKTCGLEFALYSPKNSLMPAFKNVYKRGANLNKAYIRDKGPKETGDQGQNSYKALEEYRELTWYIEVKNNDQSYNAYKNLLQLIYYHAFLPEVRENEALITDFINRTKEWNRKETEERLNTKNNKKHKNFDENDDITVNTYRYESIPDYQGESLDDYLKVLQRKQMARAKEVNEKEEGNNNYIQFIRDVVVWAFGAYLENKLKNYKNELQPPLSKENIGLNDTLKELFPEEKVKSPFNIKCRFSISTFIDNKGKSTDNTSAEAVKTDGKEDEKDKKNIKRKDLLCFYLFLRLLDENEICKLQHQFIKYRCSLKERRFPGNRTKLEKETELLAELEELMELVRFTMPSIPEISAKAESGYDTMIKKYFKDFIEKKVFKNPKTSNLYYHSDSKTPVTRKYMALLMRSAPLHLYKDIFKGYYLITKKECLEYIKLSNIIKDYQNSLNELHEQLERIKLKSEKQNGKDSLYLDKKDFYKVKEYVENLEQVARYKHLQHKINFESLYRIFRIHVDIAARMVGYTQDWERDMHFLFKALVYNGVLEERRFEAIFNNNDDNNDGRIVKKIQNNLNNKNRELVSMLCWNKKLNKNEFGAIIWKRNPIAHLNHFTQTEQNSKSSLESLINSLRILLAYDRKRQNAVTKTINDLLLNDYHIRIKWEGRVDEGQIYFNIKEKEDIENEPIIHLKHLHKKDCYIYKNSYMFDKQKEWICNGIKEEVYDKSILKCIGNLFKFDYEDKNKSSANPKHT SEQ PaludibacterMRVSKVKVKDGGKDKMVLVHRKTTGAQLVYSGQPVSNETSNILPE ID propionicigenesKKRQSFDLSTLNKTIIKFDTAKKQKLNVDQYKIVEKIFKYPKQELPK NO: C2c2 aminoQIKAEEILPFLNHKFQEPVKYWKNGKEESFNLTLLIVEAVQAQDKR 109 acid sequenceKLQPYYDWKTWYIQTKSDLLKKSIENNRIDLTENLSKRKKALLAWETEFTASGSIDLTHYHKVYMTDVLCKMLQDVKPLTDDKGKINTNAYHRGLKKALQNHQPAIFGTREVPNEANRADNQLSIYHLEVVKYLEHYFPIKTSKRRNTADDIAHYLKAQTLKTTIEKQLVNAIRANIIQQGKTNHHELKADTTSNDLIRIKTNEAFVLNLTGTCAFAANNIRNMVDNEQTNDILGKGDFIKSLLKDNTNSQLYSFFFGEGLSTNKAEKETQLWGIRGAVQQIRNNVNHYKKDALKTVFNISNFENPTITDPKQQTNYADTIYKARFINELEKIPEAFAQQLKTGGAVSYYTIENLKSLLTTFQFSLCRSTIPFAPGFKKVFNGGINYQNAKQDESFYELMLEQYLRKENFAEESYNARYFMLKLIYNNLFLPGFTTDRKAFADSVGFVQMQNKKQAEKVNPRKKEAYAFEAVRPMTAADSIADYMAYVQSELMQEQNKKEEKVAEETRINFEKFVLQVFIKGFDSFLRAKEFDFVQMPQPQLTATASNQQKADKLNQLEASITADCKLTPQYAKADDATHIAFYVFCKLLDAAHLSNLRNELIKFRESVNEFKFHHLLEIIEICLLSADVVPTDYRDLYSSEADCLARLRPFIEQGADITNWSDLFVQSDKHSPVIHANIELSVKYGTTKLLEQIINKDTQFKTTEANFTAWNTAQKSIEQLIKQREDHHEQWVKAKNADDKEKQERKREKSNFAQKFIEKHGDDYLDICDYINTYNWLDNKMHFVHLNRLHGLTIELLGRMAGFVALFDRDFQFFDEQQIADEFKLHGFVNLHSIDKKLNEVPTKKIKEIYDIRNKIIQINGNKINESVRANLIQFISSKRNYYNNAFLHVSNDEIKEKQMYDIRNHIAHFNYLTKDAADFSLIDLINELRELLHYDRKLKNAVSKAFIDLFDKHGMILKLKLNADHKLKVESLEPKKIYHLGSSAKDKPEYQYCTNQVMMAYCNMCRSLLEMKK SEQ LeptotrichiaMYMKITKIDGVSHYKKQDKGILKKKWKDLDERKQREKIEARYNKQ ID wadei (Lwa)IESKIYKEFFRLKNKKRIEKEEDQNIKSLYFFIKELYLNEKNEEWELK NO: C2c2 aminoNINLEILDDKERVIKGYKFKEDVYFFKEGYKEYYLRILFNNLIEKVQ 110 acid sequenceNENREKVRKNKEFLDLKEIFKKYKNRKIDLLLKSINNNKINLEYKKENVNEEIYGINPTNDREMTFYELLKEIIEKKDEQKSILEEKLDNFDITNFLENIEKIFNEETEINIIKGKVLNELREYIKEKEENNSDNKLKQIYNLELKKYIENNFSYKKQKSKSKNGKNDYLYLNFLKKIMFIEEVDEKKEINKEKFKNKINSNFKNLFVQHILDYGKLLYYKENDEYIKNTGQLETKDLEYIKTKETLIRKMAVLVSFAANSYYNLFGRVSGDILGTEVVKSSKTNVIKVGSHIFKEKMLNYFFDFEIFDANKIVEILESISYSIYNVRNGVGHFNKLILGKYKKKDINTNKRIEEDLNNNEEIKGYFIKKRGEIERKVKEKFLSNNLQYYYSKEKIENYFEVYEFEILKRKIPFAPNFKRIIKKGEDLFNNKNNKKYEYFKNFDKNSAEEKKEFLKTRNFLLKELYYNNFYKEFLSKKEEFEKIVLEVKEEKKSRGNINNKKSGVSFQSIDDYDTKINISDYIASIHKKEMERVEKYNEEKQKDTAKYIRDFVEEIFLTGFINYLEKDKRLHFLKEEFSILCNNNNNVVDFNININEEKIKEFLKENDSKTLNLYLFFNMIDSKRISEFRNELVKYKQFTKKRLDEEKEFLGIKIELYETLIEFVILTREKLDTKKSEEIDAWLVDKLYVKDSNEYKEYEEILKLFVDEKILSSKEAPYYATDNKTPILLSNFEKTRKYGTQSFLSEIQSNYKYSKVEKENIEDYNKKEEIEQKKKSNIEKLQDLKVELHKKWEQNKITEKEIEKYNNTTRKINEYNYLKNKEELQNVYLLHEMLSDLLARNVAFFNKWERDFKFIVIAIKQFLRENDKEKVNEFLNPPDNSKGKKVYFSVSKYKNTVENIDGIHKNFMNLIFLNNKFMNRKIDKMNCAIWVYFRNYIAHFLHLHTKNEKISLISQMNLLIKLFSYDKKVQNHILKSTKTLLEKYNIQINFEISNDKNEVFKYKIKNRLYSKKGKMLGKNNKFEILENEFLENVKAM LEYSE SEQ BergeyellaMENKTSLGNNIYYNPFKPQDKSYFAGYFNAAMENTDSVFRELGKR ID zoohelcumLKGKEYTSENFFDAIFKENISLVEYERYVKLLSDYFPMARLLDKKEV NO: Cas13bPIKERKENFKKNFKGIIKAVRDLRNFYTHKEHGEVEITDEIFGVLDE 128MLKSTVLTVKKKKVKTDKTKEILKKSIEKQLDILCQKKLEYLRDTARKIEEKRRNQRERGEKELVAPFKYSDKRDDLIAAIYNDAFDVYIDKKKDSLKESSKAKYNTKSDPQQEEGDLKIPISKNGVVFLLSLFLTKQEIHAFKSKIAGFKATVIDEATVSEATVSHGKNSICFMATHEIFSHLAYKKLKRKVRTAEINYGEAENAEQLSVYAKETLMMQMLDELSKVPDVVYQNLSEDVQKTFIEDWNEYLKENNGDVGTMEEEQVIHPVIRKRYEDKFNYFAIRFLDEFAQFPTLRFQVHLGNYLHDSRPKENLISDRRIKEKITVFGRLSELEHKKALFIKNTETNEDREHYWEIFPNPNYDFPKENISVNDKDFPIAGSILDREKQPVAGKIGIKVKLLNQQYVSEVDKAVKAHQLKQRKASKPSIQNIIEEIVPINESNPKEAIVFGGQPTAYLSMNDIHSILYEFFDKWEKKKEKLEKKGEKELRKEIGKELEKKIVGKIQAQIQQIIDKDTNAKILKPYQDGNSTAIDKEKLIKDLKQEQNILQKLKDEQTVREKEYNDFIAYQDKNREINKVRDRNHKQYLKDNLKRKYPEAPARKEVLYYREKGKVAVWLANDIKRFMPTDFKNEWKGEQHSLLQKSLAYYEQCKEELKNLLPEKVFQHLPFKLGGYFQQKYLYQFYTCYLDKRLEYISGLVQQAENFKSENKVFKKVENECFKFLKKQNYTHKELDARVQSILGYPIFLERGFMDEKPTIIKGKTFKGNEALFADWFRYYKEYQNFQTFYDTENYPLVELEKKQADRKRKTKIYQQKKNDVFTLLMAKHIFKSVFKQDSIDQFSLEDLYQSREERLGNQERARQTGERNTNYIWNKTVDLKLCDGKITVENVKLKNVGDFIKYEYDQRVQAFLKYEENIEWQAFLIKESKEEENYPYVVEREIEQYEKVRREELLKEVHLIEEYILEKVKDKEILKKGDNQNFKYYILNGLLKQLKNEDVESYKVFNLNTEPEDVNINQLKQEATDLEQKAFVLTYIRNKFAHNQLPKKEFWDYCQEKYGKIEKEKTY AEYFAEVFKKEKEALIK SEQPrevotella MEDDKKTTDSIRYELKDKHFWAAFLNLARHNVYITVNHINKILEEG ID intermediaEINRDGYETTLKNTWNEIKDINKKDRLSKLIIKHFPFLEAATYRLNPT NO: Cas13bDTTKQKEEKQAEAQSLESLRKSFFVFIYKLRDLRNHYSHYKHSKSLE 129RPKFEEGLLEKMYNIFNASIRLVKEDYQYNKDINPDEDFKHLDRTEEEFNYYFTKDNEGNITESGLLFFVSLFLEKKDAIWMQQKLRGFKDNRENKKKMTNEVFCRSRMLLPKLRLQSTQTQDWILLDMLNELIRCPKSLYERLREEDREKFRVPIEIADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKQIGDYKESHHLTHKLYGFERIQEFTKQNRPDEWRKFVKTFNSFETSKEPYIPETTPHYHLENQKIGIRFRNDNDKIWPSLKTNSEKNEKSKYKLDKSFQAEAFLSVHELLPMMFYYLLLKTENTDNDNEIETKKKENKNDKQEKHKIEEIIENKITEIYALYDTFANGEIKSIDELEEYCKGKDIEIGHLPKQMIAILKDEHKVMATEAERKQEEMLVDVQKSLESLDNQINEEIENVERKNSSLKSGKIASWLVNDMMRFQPVQKDNEGKPLNNSKANSTEYQLLQRTLAFFGSEHERLAPYFKQTKLIESSNPHPFLKDTEWEKCNNILSFYRSYLEAKKNFLESLKPEDWEKNQYFLKLKEPKTKPKTLVQGWKNGFNLPRGIFTEPIRKWFMKHRENITVAELKRVGLVAKVIPLFFSEEYKDSVQPFYNYHFNVGNINKPDEKNFLNCEERRELLRKKKDEFKKMTDKEKEENPSYLEFKSWNKFERELRLVRNQDIVTWLLCMELFNKKKIKELNVEKIYLKNINTNTTKKEKNTEEKNGEEKNIKEKNNILNRIMPMRLPIKVYGRENFSKNKKKKIRRNTFFTVYIEEKGTKLLKQGNFKALERDRRLGGLFSFVKTPSKAESKSNTISKLRVEYELGEYQKARIEIIKDMLALEKTLIDKYNSLDTDNFNKMLTDWLELKGEPDKASFQNDVDLLIAVRNAFSHNQYPMRNRIAFANINPFSLSSANTSEEKGLGIANQLKDKTHKTIEKIIEIEKPIE TKE SEQ PrevotellaMQKQDKLFVDRKKNAIFAFPKYITIMENKEKPEPIYYELTDKHFWA ID buccae Cas13bAFLNLARHNVYTTINHINRRLEIAELKDDGYMMGIKGSWNEQAKK NO:LDKKVRLRDLIMKHFPFLEAAAYEMTNSKSPNNKEQREKEQSEALS 130LNNLKNVLFIFLEKLQVLRNYYSHYKYSEESPKPIFETSLLKNMYKVFDANVRLVKRDYMHHENIDMQRDFTHLNRKKQVGRTKNIIDSPNFHYHFADKEGNMTIAGLLFFVSLFLDKKDAIWMQKKLKGFKDGRNLREQMTNEVFCRSRISLPKLKLENVQTKDWMQLDMLNELVRCPKSLYERLREKDRESFKVPFDIFSDDYNAEEEPFKNTLVRHQDRFPYFVLRYFDLNEIFEQLRFQIDLGTYHFSIYNKRIGDEDEVRHLTHEILYGFARIQDFAPQNQPEEWRKLVKDLDHFETSQEPYISKTAPHYHLENEKIGIKFCSAHNNLFPSLQTDKTCNGRSKFNLGTQFTAEAFLSVHELLPMMFYYLLLTKDYSRKESADKVEGIIRKEISNIYAIYDAFANNEINSIADLTRRLQNTNILQGHLPKQMISILKGRQKDMGKEAERKIGEMIDDTQRRLDLLCKQTNQKIRIGKRNAGLLKSGKIADWLVNDMMRFQPVQKDQNNIPINNSKANSTEYRMLQRALALFGSENFRLKAYFNQMNLVGNDNPHPFLAETQWEHQTNILSFYRNYLEARKKYLKGLKPQNWKQYQHFLILKVQKTNRNTLVTGWKNSFNLPRGIFTQPIREWFEKHNNSKRIYDQILSFDRVGFVAKAIPLYFAEEYKDNVQPFYDYPFNIGNRLKPKKRQFLDKKERVELWQKNKELFKNYPSEKKKTDLAYLDFLSWKKFERELRLIKNQDIVTWLMFKELFNMATVEGLKIGEIHLRDIDTNTANEESNNILNRIMPMKLPVKTYETDNKGNILKERPLATFYIEETETKVLKQGNFKALVKDRRLNGLFSFAETTDLNLEEHPISKLSVDLELIKYQTTRISIFEMTLGLEKKLIDKYSTLPTDSFRNMLERWLQCKANRPELKNYVNSLIAVRNAFSHNQYPMYDATLFAEVKKFTLFPSVDTKKIELNIAPQLLEIVGK AIKEIEKSENKN SEQPorphyromonas MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKFG IDgingivalis KKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQ NO: Cas13bIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHL 131EVSPDISSFITGTYSLACGRAQSRFAVFFKPDDFVLAKNRKEQLISVADGKECLTVSGFAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLDEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELRLLDPSSGHPFLSATMETAHRYTEGFYKCYLEKKREWLAKIFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKVMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVRDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPIL DPENRFFGKLLNNMSQPINDLSEQ Bacteroides MESIKNSQKSTGKTLQKDPPYFGLYLNMALLNVRKVENHIRKWLG IDpyogenes DVALLPEKSGFHSLLTTDNLSSAKWTRFYYKSRKFLPFLEMFDSDK NO: Cas13bKSYENRRETAECLDTIDRQKISSLLKEVYGKLQDIRNAFSHYHIDDQ 132SVKHTALIISSEMHRFIENAYSFALQKTRARFTGVFVETDFLQAEEKGDNKKFFAIGGNEGIKLKDNALIFLICLFLDREEAFKFLSRATGFKSTKEKGFLAVRETFCALCCRQPHERLLSVNPREALLMDMLNELNRCPDILFEMLDEKDQKSFLPLLGEEEQAHILENSLNDELCEAIDDPFEMIASLSKRVRYKNRFPYLMLRYIEEKNLLPFIRFRIDLGCLELASYPKKMGEENNYERSVTDHAMAFGRLTDFHNEDAVLQQITKGITDEVRFSLYAPRYAIYNNKIGFVRTSGSDKISFPTLKKKGGEGHCVAYTLQNTKSFGFISIYDLRKILLLSFLDKDKAKNIVSGLLEQCEKHWKDLSENLFDAIRTELQKEFPVPLIRYTLPRSKGGKLVSSKLADKQEKYESEFERRKEKLTEILSEKDFDLSQIPRRMIDEWLNVLPTSREKKLKGYVETLKLDCRERLRVFEKREKGEHPLPPRIGEMATDLAKDIIRMVIDQGVKQRITSAYYSEIQRCLAQYAGDDNRRHLDSIIRELRLKDTKNGHPFLGKVLRPGLGHTEKLYQRYFEEKKEWLEATFYPAASPKRVPRFVNPPTGKQKELPLIIRNLMKERPEWRDWKQRKNSHPIDLPSQLFENEICRLLKDKIGKEPSGKLKWNEMFKLYWDKEFPNGMQRFYRCKRRVEVFDKVVEYEYSEEGGNYKKYYEALIDEVVRQKISSSKEKSKLQVEDLTLSVRRVFKRAINEKEYQLRLLCEDDRLLFMAVRDLYDWKEAQLDLDKIDNMLGEPVSVSQVIQLEGGQPDAVIKAECKLKDVSKLMRYCYDGRVKGLMPYFANHEATQEQVEMELRHYEDHRRRVFNWVFALEKSVLKNEKLRRFYEESQGGCEHRRCIDALRKASLVSEEEYEFLVHIRNKSAHNQFPDLEIGKLPPNVTSGFCECIWSKYKAIICRIIPFIDPERRFFGKLLEQK SEQ Cas13cMTEKKSIIFKNKSSVEIVKKDIFSQTPDNMIRNYKITLKISEKNPRVVE IDAEIEDLMNSTILKDGRRSARREKSMTERKLIEEKVAENYSLLANCPM NO:EEVDSIKIYKIKRFLTYRSNMLLYFASINSFLCEGIKGKDNETEEIWH 133LKDNDVRKEKVKENFKNKLIQSTENYNSSLKNQIEEKEKLLRKESKKGAFYRTIIKKLQQERIKELSEKSLTEDCEKIIKLYSELRHPLMHYDYQYFENLFENKENSELTKNLNLDIFKSLPLVRKMKLNNKVNYLEDNDTLFVLQKTKKAKTLYQIYDALCEQKNGFNKFINDFFVSDGEENTVFKQIINEKFQSEMEFLEKRISESEKKNEKLKKKFDSMKAHFHNINSEDTKEAYFWDIHSSSNYKTKYNERKNLVNEYTELLGSSKEKKLLREEITQINRKLLKLKQEMEEITKKNSLFRLEYKMKIAFGFLFCEFDGNISKFKDEFDASNQEKIIQYHKNGEKYLTYFLKEEEKEKFNLEKMQKIIQKTEEEDWLLPETKNNLFKFYLLTYLLLPYELKGDFLGFVKKHYYDIKNVDFMDENQNNIQVSQTVEKQEDYFYHKIRLFEKNTKKYEIVKYSIVPNEKLKQYFEDLGIDIKYLTGSVESGEKWLGENLGIDIKYLTVEQKS EVSEEKIKKFL SEQ Cas13cMEKDKKGEKIDISQEMIEEDLRKILILFSRLRHSMVHYDYEFYQALY IDSGKDFVISDKNNLENRMISQLLDLNIFKELSKVKLIKDKAISNYLDK NO:NTTIHVLGQDIKAIRLLDIYRDICGSKNGFNKFINTMITISGEEDREYK 134EKVIEHFNKKMENLSTYLEKLEKQDNAKRNNKRVYNLLKQKLIEQQKLKEWFGGPYVYDIHSSKRYKELYIERKKLVDRHSKLFEEGLDEKNKKELTKINDELSKLNSEMKEMTKLNSKYRLQYKLQLAFGFILEEFDLNIDTFINNFDKDKDLIISNFMKKRDIYLNRVLDRGDNRLKNIIKEYKFRDTEDIFCNDRDNNLVKLYILMYILLPVEIRGDFLGFVKKNYYDMKHVDFIDKKDKEDKDTFFHDLRLFEKNIRKLEITDYSLSSGFLSKEHKVDIEKKINDFINRNGAMKLPEDITIEEFNKSLILPIMKNYQINFKLLNDIEISALFKIAKDRSITFKQAIDEIKNEDIKKNSKKNDKNNHKDKNINFTQLMKRALHEKIPYKAGMYQIRNNISHIDMEQLYIDPLNSYMNSNKNNITISEQIEKIIDVCVTGGVTGKELNNNIINDYYMKKEKLVFNLKLRKQNDIVSIESQEKNKREEFVFKKYGLDYKDGEINIIEVIQKVNSLQEELRNIKETSKEKLKNKETLFRDISLINGTIRKNINFKIKEMVLDIVRMDEIRHINIHIYYKGENYTRSNIIKFKYAIDGENKKYYLKQHEINDINLELKDKFVTLICNMDKHPNKNKQTINLESNYIQNVKFIIP SEQ Cas13cMENKGNNKKIDFDENYNILVAQIKEYFTKEIENYNNRIDNIIDKKEL IDLKYSEKKEESEKNKKLEELNKLKSQKLKILTDEEIKADVIKIIKIFSDL NO:RHSLMHYEYKYFENLFENKKNEELAELLNLNLFKNLTLLRQMKIEN 135KTNYLEGREEFNIIGKNIKAKEVLGHYNLLAEQKNGFNNFINSFFVQDGTENLEFKKLIDEHFVNAKKRLERNIKKSKKLEKELEKMEQHYQRLNCAYVWDIHTSTTYKKLYNKRKSLIEEYNKQINEIKDKEVITAINVELLRIKKEMEEITKSNSLFRLKYKMQIAYAFLEIEFGGNIAKFKDEFDCSKMEEVQKYLKKGVKYLKYYKDKEAQKNYEFPFEEIFENKDTHNEEWLENTSENNLFKFYILTYLLLPMEFKGDFLGVVKKHYYDIKNVDFTDESEKELSQVQLDKMIGDSFFHKIRLFEKNTKRYEIIKYSILTSDEIKRYFRLLELDVPYFEYEKGTDEIGIFNKNIILTIFKYYQIIFRLYNDLEIHGLFNISSDLDKILRDLKSYGNKNINFREFLYVIKQNNNSSTEEEYRKIWENLEAKYLRLHLLTPEKEEIKTKTKEELEKLNEISNLRNGICHLNYKEIIEEILKTEISEKNKEATLNEKIRKVINFIKENELDKVELGFNFINDFFMKKEQFMFGQIKQVKEGNSDSITTERERKEKNNKKLKETYELNCDNLSEFYETSNNLRERANSSSLLEDSAFLKKIGLYKVKNNKVNSKVKDEEKRIENIKRKLLKDSSDIMGMYKAEVVKKLKEKLILIFKHDEEKRIYVTVYDTSKAVPENISKEILVKRNNSKEEYFFEDNNKKYVTEYYTLEITETNELKVIPAKKLEGKEFKTEKNKENKLMLNNHYCFNVKIIY SEQ Cas13cMEEIKHKKNKSSIIRVIVSNYDMTGIKEIKVLYQKQGGVDTFNLKTII IDNLESGNLEIISCKPKEREKYRYEFNCKTEINTISITKKDKVLKKEIRKY NO:SLELYFKNEKKDTVVAKVTDLLKAPDKIEGERNHLRKLSSSTERKL 136LSKTLCKNYSEISKTPIEEIDSIKIYKIKRFLNYRSNFLIYFALINDFLCAGVKEDDINEVWLIQDKEHTAFLENRIEKITDYIFDKLSKDIENKKNQFEKRIKKYKTSLEELKTETLEKNKTFYIDSIKTKITNLENKITELSLYNSKESLKEDLIKIISIFTNLRHSLMHYDYKSFENLFENIENEELKNLLDLNLFKSIRMSDEFKTKNRTNYLDGTESFTIVKKHQNLKKLYTYYNNLCDKKNGFNTFINSFFVTDGIENTDFKNLIILHFEKEMEEYKKSIEYYKIKISNEKNKSKKEKLKEKIDLLQSELINMREHKNLLKQIYFFDIHNSIKYKELYSERKNLIEQYNLQINGVKDVTAINHINTKLLSLKNKMDKITKQNSLYRLKYKLKIAYSFLMIEFDGDVSKFKNNFDPTNLEKRVEYLDKKEEYLNYTAPKNKFNFAKLEEELQKIQSTSEMGADYLNVSPENNLFKFYILTYIMLPVEFKGDFLGFVKNHYYNIKNVDFMDESLLDENEVDSNKLNEKIENLKDSSFFNKIRLFEKNIKKYEIVKYSVSTQENMKEYFKQLNLDIPYLDYKSTDEIGIFNKNMILPIFKYYQNVFKLCNDIEIHALLALANKKQQNLEYAIYCCSKKNSLNYNELLKTFNRKTYQNLSFIRNKIAHLNYKELFSDLFNNELDLNTKVRCLIEFSQNNKFDQIDLGMNFINDYYMKKTRFIFNQRRLRDLNVPSKEKIIDGKRKQQNDSNNELLK KYGLSRTNIKDIFNKAWY SEQCas13c MKVRYRKQAQLDTFIIKTEIVNNDIFIKSIIEKAREKYRYSFLFDGEE IDKYHFKNKSSVEIVKNDIFSQTPDNMIRNYKITLKISEKNPRVVEAEIE NO:DLMNSTILKDGRRSARREKSMTERKLIEEKVAENYSLLANCPIEEVD 137SIKIYKIKRFLTYRSNMLLYFASINSFLCEGIKGKDNETEEIWHLKDNDVRKEKVKENFKNKLIQSTENYNSSLKNQIEEKEKLSSKEFKKGAFYRTIIKKLQQERIKELSEKSLTEDCEKIIKLYSELREIPLMHYDYQYFENLFENKENSELTKNLNLDIFKSLPLVRKMKLNNKVNYLEDNDTLFVLQKTKKAKTLYQIYDALCEQKNGFNKFINDFFVSDGEENTVFKQIINEKFQSEMEFLEKRISESEKKNEKLKKKLDSMKAHFRNINSEDTKEAYFWDIHSSRNYKTKYNERKNLVNEYTKLLGSSKEKKLLREEITKINRQLLKLKQEMEEITKKNSLFRLEYKMKIAFGFLFCEFDGNISKFKDEFDASNQEKIIQYHKNGEKYLTSFLKEEEKEKFNLEKMQKIIQKTEEEDWLLPETKNNLFKFYLLTYLLLPYELKGDFLGFVKKHYYDIKNVDFMDENQNNIQVSQTVEKQEDYFYHKIRLFEKNTKKYEIVKYSIVPNEKLKQYFEDLGIDIKYLTGSVESGEKWLGENLGIDIKYLTVEQKSEVSEEKNKKVSLKNNGMFNKTILLFVFKYYQIAFKLFNDIELYSLFFLREKSEKPFEVFLEELKDKMIGKQLNFGQLLYVVYEVLVKNKDLDKILSKKIDYRKDKSFSPEIAYLRNFLSHLNYSKFLDNFMKINTNKSDENKEVLIPSIKIQKMIQFIEKCNLQNQIDFDFNFVNDFYMRKEKMFFIQLKQIFPDINSTEKQKKSEKEEILRKRYHLINKKNEQIKDEHEAQSQLYEKILSLQKIFSCDKNNFYRRLKEEKLLFLEKQGKKKISMKEIKDKIASDISDLLGILKKEITRDIKDKLTEKFRYCEEKLLNISFYNHQDKKKEEGIRVFLIRDKNSDNFKFESILDDGSNKIFISKNGKEITIQCCDKVLETLMIEKNTLKIS SNGKIISLIPHYSYSIDVKY

The programmable nuclease can be Cas13. Sometimes the Cas13 can beCas13a, Cas13b, Cas13c, Cas13d, or Cas13e. In some cases, theprogrammable nuclease can be Mad7 or Mad2. In some cases, theprogrammable nuclease can be Cas12. Sometimes the Cas12 can be Cas12a,Cas12b, Cas12c, Cas12d (also referred to as CasY), or Cas12e. In somecases, the Cas12 can be a Cas12 variant (e.g., SEQ ID NO: 11), which isa specific protein variant within the Cas12 proteinfamily/classification. In some cases, the programmable nuclease can beCsm1, Cas9, C2c4, C2c8, C2c5, C2c10, C2c9, or CasZ. Sometimes, the Csm1can also be also called smCms1, miCms1, obCms1, or suCms1. SometimesCas13a can also be also called C2c2. Sometimes CasZ can also be calledCas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, or Cas14h. Theprogrammable nuclease can be a CRISPR-Cas (clustered regularlyinterspaced short palindromic repeats—CRISPR associated) nucleoproteincomplex with trans cleavage activity, which can be activated by bindingof a guide nucleic acid with a target nucleic acid. The CRISPR-Casnucleoprotein complex can comprise a Cas protein (also referred to as aCas nuclease) complexed with a guide nucleic acid, which can also bereferred to as CRISPR enzyme. A guide nucleic acid can be a CRISPR RNA(crRNA). Sometimes, a guide nucleic acid comprises a crRNA and atrans-activating crRNA (tracrRNA). The CRISPR/Cas system used to detecta modified target nucleic acids can comprise CRISPR RNAs (crRNAs),trans-activating crRNAs (tracrRNAs), Cas proteins, and nucleic acids ofa reporter.

The programmable nucleases described herein are capable of beingactivated when complexed with the guide nucleic acid and the targetnucleic acid (e.g., DNA). A programmable nuclease can be capable ofbeing activated when complexed with a guide nucleic acid and the targetdeoxyribonucleotide. The programmable nuclease can be activated uponbinding of the guide nucleic acid to its target nucleic acid anddegrades non-specifically nucleic acid in its environment. Theprogrammable nuclease may have trans cleavage activity once activated.In some embodiments, an activated DNA-activated programmable RNAnuclease non-specifically degrades RNA in its environment (e.g.,exhibits sequence-independent cleavage of RNA, such as RNA reporters). ADNA-activated programmable RNA nuclease can be a Cas protein (alsoreferred to, interchangeably, as a Cas nuclease). A crRNA and Casprotein can form a CRISPR/Cas enzyme. In some embodiments, theDNA-activated programmable RNA nuclease is a Type VI CRISPR enzyme.Sometimes, the programmable nuclease is a type V CRISPR-Cas system. Theprogrammable nuclease can be a type VI CRISPR-Cas system. Sometimes theprogrammable nuclease is a type III CRISPR-Cas system. In someembodiments, the DNA-activated programmable RNA nuclease is Cas13.Sometimes the Cas13 is Cas13a, Cas13b, Cas13c, Cas13d, or Cas13e. Insome cases, the DNA-activated programmable RNA nuclease is from at leastone of Leptotrichia shahii (Lsh), Listeria seeligeri (Lse), Leptotrichiabuccalis (Lbu), Leptotrichia wadeu (Lwa), Rhodobacter capsulatus (Rca),Herbinix hemicellulosilytica (Hhe), Paludibacter propionicigenes (Ppr),Lachnospiraceae bacterium (Lba), [Eubacterium] rectale (Ere), Listerianewyorkensis (Lny), Clostridium aminophilum (Cam), Prevotella sp. (Psm),Capnocytophaga canimorsus (Cca, Lachnospiraceae bacterium (Lba),Bergeyella zoohelcum (Bzo), Prevotella intermedia (Pin), Prevotellabuccae (Pbu), Alistipes sp. (Asp), Riemerella anatipestifer (Ran),Prevotella aurantiaca (Pau), Prevotella saccharolytica (Psa), Prevotellaintermedia (Pin2), Capnocytophaga canimorsus (Cca), Porphyromonas gulae(Pgu), Prevotella sp. (Psp), Porphyromonas gingivalis (Pig), Prevotellaintermedia (Pin3), Enterococcus italicus (Ei), Lactobacillus salivarius(Ls), or Thermus thermophilus (Tt). Sometimes the DNA-activatedprogrammable RNA nuclease is at least one of LbuCas13a, LwaCas13a,LbaCas13a, HheCas13a, PprCas13a, EreCas13a, CamCas13a, or LshCas13a.

In some embodiments, a programmable nuclease is capable of beingactivated by a target RNA to initiate trans cleavage of an RNA reporterand is capable of being activated by a target DNA to initiate transcleavage of an RNA reporter, such as a Type VI CRISPR protein (e.g.,Cas13). For example, Cas13a of the present disclosure can be activatedby a target RNA to initiate trans cleavage activity of the Cas13a forthe cleavage of an RNA reporter and can be activated by a target DNA toinitiate trans cleavage activity of the Cas13a for trans cleavage of anRNA reporter.

The trans cleavage activity of the DNA-activated programmable RNAnuclease can be activated when the crRNA is complexed with the targetdeoxyribonucleic acid. The trans cleavage activity of the DNA-activatedprogrammable RNA nuclease can be activated when the guide nucleic acidcomprising a tracrRNA and crRNA are complexed with the targetdeoxyribonucleic acid. The target deoxyribonucleic acid can be a DNA orreverse transcribed RNA, or an amplicon thereof. Preferably, the targetdeoxyribonucleic acid is single-stranded DNA. Thus, a Cas13a nuclease ofthe present disclosure can be activated by a target DNA to initiatetrans cleavage activity of the Cas13a nuclease that cleaves an RNAreporter. For example, Cas13a nucleases disclosed herein are activatedby the binding of the guide nucleic acid to a target DNA that wasreverse transcribed from an RNA to cleave nucleic acids of a reporter ina sequence-independent manner. For example, Cas13a nucleases disclosedherein are activated by the binding of the guide nucleic acid to atarget DNA that was amplified from a DNA to trans-collaterally cleavereporter molecules. The reporters can be RNA reporters. In someembodiments, the Cas13a recognizes and detects ssDNA and, further, transcleaves RNA reporters. Multiple Cas13a isolates can recognize, beactivated by, and detect target DNA as described herein, includingssDNA. For example, trans-collateral cleavage of RNA reporters can beactivated in LbuCas13a or LwaCas13a by target DNA. Therefore, aDNA-activated programmable RNA nuclease can be used to detect target DNAby assaying for cleaved RNA reporters.

In some embodiments, the programmable nuclease may be present in thecleavage reaction at a concentration of about 10 nM, about 20 nM, about30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM,about 90 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM,about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM,about 1 μM, about 10 μM, or about 100 μM. In some embodiments, theprogrammable nuclease may be present in the cleavage reaction at aconcentration of from 10 nM to 20 nM, from 20 nM to 30 nM, from 30 nM to40 nM, from 40 nM to 50 nM, from 50 nM to 60 nM, from 60 nM to 70 nM,from 70 nM to 80 nM, from 80 nM to 90 nM, from 90 nM to 100 nM, from 100nM to 200 nM, from 200 nM to 300 nM, from 300 nM to 400 nM, from 400 nMto 500 nM, from 500 nM to 600 nM, from 600 nM to 700 nM, from 700 nM to800 nM, from 800 nM to 900 nM, from 900 nM to 1 μM, from 1 μM to 10 μM,from 10 μM to 100 μM, from 10 nM to 100 nM, from 10 nM to 1 μM, from 10nM to 10 μM, from 10 nM to 100 μM, from 100 nM to 1 μM, from 100 nM to10 μM, from 100 nM to 100 μM, or from 1 μM to 100 μM. In someembodiments, the programmable nuclease may be present in the cleavagereaction at a concentration of from 20 nM to 50 μM, from 50 nM to 20 μM,or from 200 nM to 5 μM.

A DNA-activated programmable RNA nuclease can be used to detect DNA atmultiple pH values. A DNA-activated programmable RNA nuclease can beused to detect DNA at multiple pH values compared to an RNA-activatedprogrammable RNA nuclease, such as a Cas13a complexed with a guide RNAthat detects a target ribonucleic acid. For example, a Cas13 proteinthat detects a target RNA may exhibit high cleavage activity at pHvalues from 7.9 to 8.2. A Cas13 protein that detects a target DNA canexhibit consistent cleavage across a wide range of pH conditions, suchas from a pH of 6.8 to a pH of 8.2. In some embodiments, Cas13 ssDNAdetection may exhibit high cleavage activity at pH values from 6 to 6.5,from 6.1 to 6.6, from 6.2 to 6.7, from 6.3 to 6.8, from 6.4 to 6.9, from6.5 to 7, from 6.6 to 7.1, from 6.7 to 7.2, from 6.8 to 7.3, from 6.9 to7.4, from 7 to 7.5, from 7.1 to 7.6, from 7.2 to 7.7, from 7.3 to 7.8,from 7.4 to 7.9, from 7.5 to 8, from 7.6 to 8.1, from 7.7 to 8.2, from7.8 to 8.3, from 7.9 to 8.4, from 8 to 8.5, from 8.1 to 8.6, from 8.2 to8.7, from 8.3 to 8.8, from 8.4 to 8.9, from 8.5 to 9, from 6 to 8, from6.5 to 8, or from 7 to 8. Preferrably, Cas13 ssDNA detection may exhibithigh cleavage activity at pH values from 7.0 to 8.0. More preferably,Cas13 ssDNA detection may exhibit high cleavage activity at pH 7.5.

In some embodiments, a programmable nuclease is capable of beingactivated by a target RNA to initiate trans cleavage of an RNA reporterand is capable of being activated by a target DNA to initiate transcleavage of an RNA reporter, such as a Type VI CRISPR protein (e.g.,Cas13). For example, Cas13a of the present disclosure can be activatedby a target RNA to initiate trans cleavage activity of the Cas13a forthe cleavage of an RNA reporter and can be activated by a target DNA toinitiate trans cleavage activity of the Cas13a for trans cleavage of anRNA reporter. In some embodiments, target DNA binding preferences of aDNA-activated programmable RNA nuclease can be distinct from target RNAbinding preferences of a RNA-activated programmable RNA nuclease. Insome embodiments, target DNA binding preferences of a guide nucleic acidcomplexed with a DNA-activated programmable RNA nuclease can be distinctfrom target RNA binding preferences of a guide nucleic acid complexedwith a RNA-activated programmable RNA nuclease. For example, guide RNA(gRNA) binding to a target DNA, and preferably a target ssDNA, may notnecessarily correlate with the binding of the same gRNAs binding to atarget RNA. For example, gRNAs can perform at a high level regardless oftarget nucleotide identity at a 3′ position in a sequence of a targetRNA. In some embodiments, gRNAs can perform at a high level in theabsence of a G at a 3′ position in a sequence of a target DNA.Furthermore, target DNA detected by a DNA-activated programmable RNAnuclease complexed with a guide nucleic acid as disclosed herein can bedirectly from organisms, or can be indirectly generated by nucleic acidamplification methods, such as PCR and LAMP of DNA or reversetranscription of RNA. Key steps for the sensitive detection of directDNA by a DNA-activated programmable RNA nuclease, such as a Cas13a, caninclude: (1) production or isolation of DNA to concentrations aboveabout 0.1 nM per reaction for in vitro diagnostics, (2) selection of atarget DNA with the appropriate sequence features to enable DNAdetection as these some of these features are distinct from thoserequired for target RNA detection, and (3) buffer composition thatenhances DNA detection. The detection of DNA by a DNA-activatedprogrammable RNA nuclease can be connected to a variety of readoutsincluding fluorescence, lateral flow, electrochemistry, or any otherreadouts described herein. Multiplexing of a DNA-activated programmableRNA nuclease with a DNA-activated programmable DNA nuclease with RNA andDNA FQ-reporter molecules (each with a different color fluorophore),respectively, can enable detection of ssDNA or a combination of ssDNAand dsDNA, respectively. Multiplexing of different DNA-activatedprogrammable RNA nuclease that have distinct RNA reporter cleavagepreferences can enable additional multiplexing, such a firstDNA-activated programmable RNA nuclease that preferentially cleavesuracil in an RNA reporter and a second DNA-activated programmable RNAnuclease that preferentially cleaves adenines in an RNA reporter.Methods for the generation of ssDNA for a DNA-activated programmable RNAnuclease-based detection or diagnostics can include (1) asymmetric PCR,(2) asymmetric isothermal amplification, such as RPA, LAMP, SDA, etc.(3) NEAR for the production of short ssDNA molecules, and (4) conversionof RNA targets into ssDNA by a reverse transcriptase followed by RNase Hdigestion. Thus, a DNA-activated programmable RNA nuclease detection oftarget DNA is compatible with the various systems, kits, compositions,reagents, and methods disclosed herein. Cas13a DNA detection can beemployed in a DETECTR assay disclosed herein to provide CRISPRdiagnostics leveraging Type VI systems (e.g., Cas13) for the detectionof a target DNA.

Some programmable nucleases can exhibit a high turnover rate. Turnoverrate quantifies how many molecules of a detector nucleic acid eachprogrammable nuclease is cleaving per minute. Programmable nucleaseswith a higher turnover rate are more efficient and transcollateralcleavage in the DETECTR assay methods disclosed herein.

Turnover rate is quantified as the max transcleaving velocity (max slopein a plot of signal versus time in a DETECTR assay) divided by theamount of programmable nuclease complexed with the guide nucleic acidpresent in the DETECTR assay, wherein the programmable nuclease is atsaturation with respect to its active site for transcollateral cleavageof detector nucleic acids.

Turnover rate can be quantified with the following equation:

${{Turnover}\mspace{14mu}{rate}} = \frac{\begin{matrix}{{maximum}\mspace{14mu}{transcleaving}\mspace{14mu}{velocity}\mspace{14mu}{\left( \frac{AU}{\min} \right)/}} \\{{signal}\mspace{14mu}{normalization}\mspace{14mu}{factor}\mspace{14mu}\left( \frac{AU}{nM} \right)}\end{matrix}}{\begin{matrix}{{concentration}\mspace{14mu}{of}\mspace{14mu}{programmanble}\mspace{14mu}{nuclease}\mspace{14mu}{complexed}} \\{{with}\mspace{14mu}{guide}\mspace{14mu}{nucleic}\mspace{14mu}{acid}\mspace{14mu}({nM})}\end{matrix}}$

Signal normalization factor is based on a standard curve and is theamount of signal produced from a known quantity of detector nucleic acid(substrate of transcollateral cleavage). The turnover rate is, thus,expressed as cleaved detector nucleic acid molecules per minute dividedby the concentration of the programmable nuclease complexed with guidenucleic acid (can also be referred to as “nucleoprotein” or“ribonucleoprotein”). Therefore, a programmable nuclease with a highturnover rate exhibits superior and highly efficient transcollateralcleavage of detector nucleic acids in the DETECTR assay methodsdisclosed herein. For example, a programmable nuclease having at least60% sequence identity to SEQ ID NO: 11 can exhibit high a turnover rateof at least about 0.1 cleaved detector molecules per minute. Aprogrammable nuclease having a sequence of SEQ ID NO: 11 exhibits aturnover rate of at least about 0.1 cleaved detector molecules perminute. For example, a programmable nuclease (e.g., SEQ ID NO: 11) thatrecognizes a PAM of YYN complexed with a guide nucleic acid comprises aturnover rate of at least about 0.1 cleaved detector molecules perminute. The programmable nuclease may be a Type V programmable nuclease.The programmable nuclease may be a Cas12 programmable nuclease. Aprogrammable nuclease having a sequence of SEQ ID NO: 11 exhibits aturnover rate that is higher than the turnover rate of SEQ ID NO: 1. Forexample, a programmable nuclease having a sequence of SEQ ID NO: 11 canexhibit a turnover rate that is at least about 2-fold higher than theturnover rate of SEQ ID NO: 1. In some embodiments, a programmablenuclease having a sequence of SEQ ID NO: 11 can exhibit a turnover ratethat is at about 2-fold higher than the turnover rate of SEQ ID NO: 1.In some embodiments, a programmable nuclease having a sequence of SEQ IDNO: 11 complexed with a guide nucleic acid can exhibit a turnover ratethat is at least about 2-fold higher than the turnover rate of SEQ IDNO: 1 complexed with a guide nucleic acid. Thus, a programmable nucleaseof SEQ ID NO: 11 is superior and more efficient at transcollateralcleavage in the DETECTR assay methods disclosed herein than aprogrammable nuclease of SEQ ID NO: 1.

In some embodiments, programmable nucleases with a high turnover ratehave a turnover rate of at least about 0.1 cleaved detector moleculesper minute. In some embodiments, programmable nucleases with a highturnover rate have a turnover rate of at least about 0.5 cleaveddetector molecules per minute. In some embodiments, programmablenucleases with a high turnover rate have a turnover rate of at leastabout 1 cleaved detector molecules per minute. In some embodiments,programmable nucleases with a high turnover rate have a turnover rate ofat least about 2 cleaved detector molecules per minute. In someembodiments, programmable nucleases with a high turnover rate have aturnover rate of at least about 3 cleaved detector molecules per minute.In some embodiments, programmable nucleases with a high turnover ratehave a turnover rate of at least about 4 cleaved detector molecules perminute. In some embodiments, programmable nucleases with a high turnoverrate have a turnover rate of at least about 5 cleaved detector moleculesper minute. In some embodiments, programmable nucleases with a highturnover rate have a turnover rate of at least about 10 cleaved detectormolecules per minute. In some embodiments, programmable nucleases with ahigh turnover rate have a turnover rate of at least about 15 cleaveddetector molecules per minute. In some embodiments, programmablenucleases with a high turnover rate have a turnover rate of at leastabout 20 cleaved detector molecules per minute. In some embodiments,programmable nucleases with a high turnover rate have a turnover rate ofat least about 25 cleaved detector molecules per minute. In someembodiments, programmable nucleases with a high turnover rate have aturnover rate of at least about 30 cleaved detector molecules perminute. In some embodiments, programmable nucleases with a high turnoverrate have a turnover rate of at least about 35 cleaved detectormolecules per minute. In some embodiments, programmable nucleases with ahigh turnover rate have a turnover rate of at least about 40 cleaveddetector molecules per minute. In some embodiments, programmablenucleases with a high turnover rate have a turnover rate of at leastabout 45 cleaved detector molecules per minute. In some embodiments,programmable nucleases with a high turnover rate have a turnover rate ofat least about 50 cleaved detector molecules per minute. In someembodiments, programmable nucleases with a high turnover rate have aturnover rate of at least about 60 cleaved detector molecules perminute. In some embodiments, programmable nucleases with a high turnoverrate have a turnover rate of at least about 70 cleaved detectormolecules per minute. In some embodiments, programmable nucleases with ahigh turnover rate have a turnover rate of at least about 80 cleaveddetector molecules per minute. In some embodiments, programmablenucleases with a high turnover rate have a turnover rate of at leastabout 90 cleaved detector molecules per minute. In some embodiments,programmable nucleases with a high turnover rate have a turnover rate ofat least about 100 cleaved detector molecules per minute. In someembodiments, programmable nucleases with a high turnover rate have aturnover rate of at least about 0.1 to 0.5 cleaved detector moleculesper minute. In some embodiments, programmable nucleases with a highturnover rate have a turnover rate of at least about 0.5 to 1 cleaveddetector molecules per minute. In some embodiments, programmablenucleases with a high turnover rate have a turnover rate of at leastabout 1 to 1.5 cleaved detector molecules per minute. In someembodiments, programmable nucleases with a high turnover rate have aturnover rate of at least about 1.5 to 2 cleaved detector molecules perminute. In some embodiments, programmable nucleases with a high turnoverrate have a turnover rate of at least about 2 to 2.5 cleaved detectormolecules per minute. In some embodiments, programmable nucleases with ahigh turnover rate have a turnover rate of at least about 2.5 to 3cleaved detector molecules per minute. In some embodiments, programmablenucleases with a high turnover rate have a turnover rate of at leastabout 3 to 3.5 cleaved detector molecules per minute. In someembodiments, programmable nucleases with a high turnover rate have aturnover rate of at least about 3.5 to 4 cleaved detector molecules perminute. In some embodiments, programmable nucleases with a high turnoverrate have a turnover rate of at least about 4 to 4.5 cleaved detectormolecules per minute. In some embodiments, programmable nucleases with ahigh turnover rate have a turnover rate of at least about 4.5 to 5cleaved detector molecules per minute. In some embodiments, programmablenucleases with a high turnover rate have a turnover rate of at leastabout 5 to 10 cleaved detector molecules per minute. In someembodiments, programmable nucleases with a high turnover rate have aturnover rate of at least about 10 to 15 cleaved detector molecules perminute. In some embodiments, programmable nucleases with a high turnoverrate have a turnover rate of at least about 15 to 20 cleaved detectormolecules per minute. In some embodiments, programmable nucleases with ahigh turnover rate have a turnover rate of at least about 20 to 25cleaved detector molecules per minute. In some embodiments, programmablenucleases with a high turnover rate have a turnover rate of at leastabout 25 to 30 cleaved detector molecules per minute. In someembodiments, programmable nucleases with a high turnover rate have aturnover rate of at least about 30 to 35 cleaved detector molecules perminute. In some embodiments, programmable nucleases with a high turnoverrate have a turnover rate of at least about 35 to 40 cleaved detectormolecules per minute. In some embodiments, programmable nucleases with ahigh turnover rate have a turnover rate of at least about 40 to 45cleaved detector molecules per minute. In some embodiments, programmablenucleases with a high turnover rate have a turnover rate of at leastabout 45 to 50 cleaved detector molecules per minute. In someembodiments, programmable nucleases with a high turnover rate have aturnover rate of at least about 50 to 60 cleaved detector molecules perminute. In some embodiments, programmable nucleases with a high turnoverrate have a turnover rate of at least about 60 to 70 cleaved detectormolecules per minute. In some embodiments, programmable nucleases with ahigh turnover rate have a turnover rate of at least about 70 to 80cleaved detector molecules per minute. In some embodiments, programmablenucleases with a high turnover rate have a turnover rate of at leastabout 80 to 90 cleaved detector molecules per minute. In someembodiments, programmable nucleases with a high turnover rate have aturnover rate of at least about 90 to 100 cleaved detector molecules perminute.

Guide Nucleic Acids

The reagents of this disclosure may comprise a guide nucleic acid. Theguide nucleic acid can bind to a single stranded target nucleic acid orportion thereof as described herein. For example, the guide nucleic acidcan bind to a target nucleic acid such as nucleic acid from a virus or abacterium or other agents responsible for a disease, or an ampliconthereof, as described herein. The guide nucleic acid can bind to atarget nucleic acid such as a nucleic acid from a bacterium, a virus, aparasite, a protozoa, a fungus or other agents responsible for adisease, or an amplicon thereof, as described herein and furthercomprising a mutation, such as a single nucleotide polymorphism (SNP),which can confer resistance to a treatment, such as antibiotictreatment. The guide nucleic acid can bind to a target nucleic acid suchas a nucleic acid, preferably DNA, from a cancer gene or gene associatedwith a genetic disorder, or an amplicon thereof, as described herein.The guide nucleic acid comprises a segment of nucleic acids that arereverse complementary to the target nucleic acid. Often the guidenucleic acid binds specifically to the target nucleic acid. The targetnucleic acid may be a reversed transcribed RNA, DNA, DNA amplicon, orsynthetic nucleic acids. The target nucleic acid can be asingle-stranded DNA or DNA amplicon of a nucleic acid of interest. Aguide nucleic acid may be a non-naturally occurring guide nucleic acid.A non-naturally occurring guide nucleic acid may comprise an engineeredsequence having a repeat and a spacer that hybridizes to a targetnucleic acid sequence of interest. A non-naturally occurring guidenucleic acid may be recombinantly expressed or chemically synthezised.

A guide nucleic acid (gRNA) sequence (e.g., a non-naturally occurringgRNA) may hybridize to a target sequence of a target nucleic acid. Theterm “gRNA” may be used interchangeably with the term “crRNA.” A gRNAcomprises a repeat region corresponding to a specific programmablenuclease (e.g., a Cas protein), for example the repeat sequencesprovided in TABLE 30. In some embodiments, the repeat region maycomprise mutations or truncations with respect to the repeat sequencesin pre-crRNA. The repeat sequence interacts with the programmablenuclease (e.g., a Cas protein), allowing for the gRNA and theprogrammable nuclease to form a complex. This complex may be referred toas a nucleoprotein. A spacer sequence may be positioned 3′ of the repeatregion. The spacer sequence may hybridize to a target sequence of thetarget nucleic acid, where the target sequence is a segment of a targetnucleic acid. The spacer sequences may be reverse complementary to thetarget sequence. In some cases, the spacer sequence may be sufficientlyreverse complementary to a target sequence to allow for hybridization,however, may not necessarily be 100% reverse complementary. In someembodiments, a programmable nuclease (e.g., a Cas protein) may cleave aprecursor RNA (“pre-crRNA”) to produce a gRNA, also referred to as a“mature guide RNA.” A programmable nuclease (e.g., a Cas protein) thatcleaves pre-crRNA to produce a mature guide RNA is said to havepre-crRNA processing activity.

A guide nucleic acid can comprise a sequence that is, at least in part,reverse complementary to the sequence of a target nucleic acid. Theguide nucleic acid may be a non-naturally occurring guide nucleic acid.A non-naturally occurring guide nucleic acid may comprise an engineeredsequence having a repeat and a spacer that hybridizes to a targetnucleic acid sequence of interest. A non-naturally occurring guidenucleic acid may be recombinantly expressed or chemically synthezised. Aguide nucleic acid can be a crRNA. Sometimes, a guide nucleic acidcomprises a crRNA and tracrRNA. The guide nucleic acid can bindspecifically to the target nucleic acid. In some cases, the guidenucleic acid is not naturally occurring and made by artificialcombination of otherwise separate segments of sequence. Often, theartificial combination is performed by chemical synthesis, by geneticengineering techniques, or by the artificial manipulation of isolatedsegments of nucleic acids. In some cases, the segment of a guide nucleicacid that comprises a sequence that is reverse complementary to thetarget nucleic acid is 20 nucleotides in length. A guide nucleic acidcan have at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 nucleotides reverse complementary to atarget nucleic acid. In some cases, the guide nucleic acid can be 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 nucleotides in length. For example, a guide nucleic acid maybe at least 10 bases. In some embodiments, a guide nucleic acid may befrom 10 to 50 bases. In some embodiments, a guide nucleic acid may be atleast 25 bases. In some cases, the guide nucleic acid has from exactlyor about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt,from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, fromabout 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 ntto about 25 nt, from about 19 nt to about 30 nt, from about 19 nt toabout 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt,from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, fromabout 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20nt to about 60 nt reverse complementary to a target nucleic acid. Insome cases, the guide nucleic acid has from about 10 nt to about 60 nt,from about 20 nt to about 50 nt, or from about 30 nt to about 40 ntreverse complementary to a target nucleic acid. It is understood thatthe sequence of a guide nucleic acid need not be 100% reversecomplementary to that of its target nucleic acid to be specificallyhybridizable, hybridizable, or bind specifically. The guide nucleic acidcan have a sequence comprising at least one uracil in a region fromnucleic acid residue 5 to 20 that is reverse complementary to amodification variable region in the target nucleic acid. The guidenucleic acid, in some cases, has a sequence comprising at least oneuracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to20 that is reverse complementary to a modification variable region inthe target nucleic acid. The guide nucleic acid can have a sequencecomprising at least one uracil in a region from nucleic acid residue 5to 20 that is reverse complementary to a methylation variable region inthe target nucleic acid. The guide nucleic acid, in some cases, has asequence comprising at least one uracil in a region from nucleic acidresidue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to amethylation variable region in the target nucleic acid. The guidenucleic acid can hybridize with a target nucleic acid.

The guide nucleic acid (e.g., a non-naturally occurring guide nucleicacid) can be selected from a group of guide nucleic acids that have beentiled against the nucleic acid sequence of a strain of an infection orgenomic locus of interest. The guide nucleic acid can be selected from agroup of guide nucleic acids that have been tiled against the nucleicacid sequence of a strain of HPV 16 or HPV18. Often, guide nucleic acidsthat are tiled against the nucleic acid of a strain of an infection orgenomic locus of interest can be pooled for use in a method describedherein. Often, these guide nucleic acids are pooled for detecting atarget nucleic acid in a single assay. The pooling of guide nucleicacids that are tiled against a single target nucleic acid can enhancethe detection of the target nucleic using the methods described herein.The pooling of guide nucleic acids that are tiled against a singletarget nucleic acid can ensure broad coverage of the target nucleic acidwithin a single reaction using the methods described herein. The tiling,for example, is sequential along the target nucleic acid. Sometimes, thetiling is overlapping along the target nucleic acid. In some instances,the tiling comprises gaps between the tiled guide nucleic acids alongthe target nucleic acid. In some instances, the tiling of the guidenucleic acids is non-sequential. Often, a method for detecting a targetnucleic acid comprises contacting a target nucleic acid to a pool ofguide nucleic acids and a programmable nuclease, wherein a guide nucleicacid sequence of the pool of guide nucleic acids has a sequence selectedfrom a group of tiled guide nucleic acid that correspond to nucleic acidsequence of a target nucleic acid; and assaying for a signal produce bycleavage of at least some nucleic acids of a reporter of a population ofnucleic acids of a reporter. Pooling of guide nucleic acids can ensurebroad spectrum identification, or broad coverage, of a target specieswithin a single reaction. This can be particularly helpful in diseasesor indications, like sepsis, that may be caused by multiple organisms.

Reporters

Described herein are reagents comprising a reporter. The reporter cancomprise a single stranded nucleic acid and a detection moiety, whereinthe nucleic acid is capable of being cleaved by the activatedprogrammable nuclease, releasing the detection moiety, and, generating adetectable signal. As used herein, “reporter” is used interchangeablywith “detector nucleic acid” or “reporter molecule”. The programmablenucleases disclosed herein, activated upon hybridization of a guide RNAto a target nucleic acid, can cleave the reporter. Cleaving the“reporter” may be referred to herein as cleaving the “detector nucleicacid,” the “reporter molecule,” or the “nucleic acid of the reporter.”

A major advantage of the compositions and methods disclosed herein isthe design of excess reporters to total nucleic acids in an unamplifiedor an amplified sample, not including the nucleic acid of the reporter.Total nucleic acids can include the target nucleic acids and non-targetnucleic acids, not including the nucleic acid of the reporter. Thenon-target nucleic acids can be from the original sample, either lysedor unlysed. The non-target nucleic acids can also be byproducts ofamplification. Thus, the non-target nucleic acids can include bothnon-target nucleic acids from the original sample, lysed or unlysed, andfrom an amplified sample. The presence of a large amount of non-targetnucleic acids, an activated programmable nuclease may be inhibited inits ability to bind and cleave the reporter sequences. This is becausethe activated programmable nucleases collaterally cleaves any nucleicacids. If total nucleic acids are in present in large amounts, they mayoutcompete reporters for the programmable nucleases. The compositionsand methods disclosed herein are designed to have an excess of reporterto total nucleic acids, such that the detectable signals from DETECTRreactions are particularly superior. In some embodiments, the reportercan be present in at least 1.5 fold, at least 2 fold, at least 3 fold,at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, atleast 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, atleast 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, atleast 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, atleast 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, atleast 100 fold, from 1.5 fold to 100 fold, from 2 fold to 10 fold, from10 fold to 20 fold, from 20 fold to 30 fold, from 30 fold to 40 fold,from 40 fold to 50 fold, from 50 fold to 60 fold, from 60 fold to 70fold, from 70 fold to 80 fold, from 80 fold to 90 fold, from 90 fold to100 fold, from 1.5 fold to 10 fold, from 1.5 fold to 20 fold, from 10fold to 40 fold, from 20 fold to 60 fold, or from 10 fold to 80 foldexcess of total nucleic acids.

A second significant advantage of the compositions and methods disclosedherein is the design of an excess volume comprising the guide nucleicacid, the programmable nuclease, and the reporter, which contacts asmaller volume comprising the sample with the target nucleic acid ofinterest. The smaller volume comprising the sample can be unlysedsample, lysed sample, or lysed sample which has undergone anycombination of reverse transcription, amplification, and in vitrotranscription. The presence of various reagents in a crude, non-lysedsample, a lysed sample, or a lysed and amplified sample, such as buffer,magnesium sulfate, salts, the pH, a reducing agent, primers, dNTPs,NTPs, cellular lysates, non-target nucleic acids, primers, or othercomponents, can inhibit the ability of the programmable nuclease tobecome activated or to find and cleave the nucleic acid of the reporter.This may be due to nucleic acids that are not the reporter outcompetingthe nucleic acid of the reporter, for the programmable nuclease.Alternatively, various reagents in the sample may simply inhibit theactivity of the programmable nuclease. Thus, the compositions andmethods provided herein for contacting an excess volume comprising theguide nucleic acid, the programmable nuclease, and the reporter to asmaller volume comprising the sample with the target nucleic acid ofinterest provides for superior detection of the target nucleic acid byensuring that the programmable nuclease is able to find and cleaves thenucleic acid of the reporter. In some embodiments, the volume comprisingthe guide nucleic acid, the programmable nuclease, and the reporter (canbe referred to as “a second volume”) is 4-fold greater than a volumecomprising the sample (can be referred to as “a first volume”). In someembodiments, the volume comprising the guide nucleic acid, theprogrammable nuclease, and the reporter (can be referred to as “a secondvolume”) is at least 1.5 fold, at least 2 fold, at least 3 fold, atleast 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, atleast 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, atleast 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, atleast 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, atleast 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, atleast 100 fold, from 1.5 fold to 100 fold, from 2 fold to 10 fold, from10 fold to 20 fold, from 20 fold to 30 fold, from 30 fold to 40 fold,from 40 fold to 50 fold, from 50 fold to 60 fold, from 60 fold to 70fold, from 70 fold to 80 fold, from 80 fold to 90 fold, from 90 fold to100 fold, from 1.5 fold to 10 fold, from 1.5 fold to 20 fold, from 10fold to 40 fold, from 20 fold to 60 fold, or from 10 fold to 80 foldgreater than a volume comprising the sample (can be referred to as “afirst volume”). In some embodiments, the volume comprising the sample isat least 0.5 μL, at least 1 μL, at least at least 1 μL, at least 2 μL,at least 3 μL, at least 4 μL, at least 5 μL, at least 6 μL, at least 7μL, at least 8 μL, at least 9 μL, at least 10 μL, at least 11 μL, atleast 12 μL, at least 13 μL, at least 14 μL, at least 15 μL, at least 16μL, at least 17 μL, at least 18 μL, at least 19 μL, at least 20 μL, atleast 25 μL, at least 30 μL, at least 35 μL, at least 40 μL, at least 45μL, at least 50 μL, at least 55 μL, at least 60 μL, at least 65 μL, atleast 70 μL, at least 75 μL, at least 80 μL, at least 85 μL, at least 90μL, at least 95 μL, at least 100 μL, from 0.5 μL to 5 μL μL, from 5 μLto 10 μL, from 10 μL to 15 μL, from 15 μL to 20 μL, from 20 μL to 25 μL,from 25 μL to 30 μL, from 30 μL to 35 μL, from 35 μL to 40 μL, from 40μL to 45 μL, from 45 μL to 50 μL, from 10 μL to 20 μL, from 5 μL to 20μL, from 1 μL to 40 μL, from 2 μL to 10 μL, or from 1 μL to 10 μL. Insome embodiments, the volume comprising the programmable nuclease, theguide nucleic acid, and the reporter is at least 10 μL, at least 11 μL,at least 12 μL, at least 13 μL, at least 14 μL, at least 15 μL, at least16 μL, at least 17 μL, at least 18 μL, at least 19 μL, at least 20 μL,at least 21 μL, at least 22 μL, at least 23 μL, at least 24 μL, at least25 μL, at least 26 μL, at least 27 μL, at least 28 μL, at least 29 μL,at least 30 μL, at least 40 μL, at least 50 μL, at least 60 μL, at least70 μL, at least 80 μL, at least 90 μL, at least 100 μL, at least 150 μL,at least 200 μL, at least 250 μL, at least 300 μL, at least 350 μL, atleast 400 μL, at least 450 μL, at least 500 μL, from 10 μL to 15 μL μL,from 15 μL to 20 μL, from 20 μL to 25 μL, from 25 μL to 30 μL, from 30μL to 35 μL, from 35 μL to 40 μL, from 40 μL to 45 μL, from 45 μL to 50μL, from 50 μL to 55 μL, from 55 μL to 60 μL, from 60 μL to 65 μL, from65 μL to 70 μL, from 70 μL to 75 μL, from 75 μL to 80 μL, from 80 μL to85 μL, from 85 μL to 90 μL, from 90 μL to 95 μL, from 95 μL to 100 μL,from 100 μL to 150 μL, from 150 μL to 200 μL, from 200 μL to 250 μL,from 250 μL to 300 μL, from 300 μL to 350 μL, from 350 μL to 400 μL,from 400 μL to 450 μL, from 450 μL to 500 μL, from 10 μL to 20 μL, from10 μL to 30 μL, from 25 μL to 35 μL, from 10 μL to 40 μL, from 20 μL to50 μL, from 18 μL to 28 μL, or from 17 μL to 22 μL.

As described herein, nucleic acid sequences comprising DNA may bedetected using a DNA-activated programmable RNA nuclease, aDNA-activated programmable DNA nuclease, an RNA-activated programmableRNA nuclease, or any combination thereof, and other reagents disclosedherein. The DNA-activated programmable RNA nuclease may be activated andcleave an RNA reporter upon binding of a guide nucleic acid to a targetDNA. In some cases, the reporter comprises a nucleic acid, which is asingle-stranded nucleic acid sequence comprising ribonucleotides.Additionally, detection by a DNA-activated programmable RNA nuclease,which can cleave RNA reporters, allows for multiplexing with aDNA-activated programmable DNA nuclease that can cleave DNA reporters(e.g., Type V CRISPR enzyme). In some cases, the nucleic acid of areporter is a single-stranded nucleic acid sequence comprisingdeoxyribonucleotides.

The nucleic acid of a reporter can be a single-stranded nucleic acidsequence comprising at least one deoxyribonucleotide and at least oneribonucleotide. In some cases, the nucleic acid of a reporter is asingle-stranded nucleic acid comprising at least one ribonucleotideresidue at an internal position that functions as a cleavage site. Insome cases, the nucleic acid of a reporter comprises at least 2, 3, 4,5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position. Insome cases, the nucleic acid of a reporter comprises from 2 to 10, from3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at aninternal position. Sometimes the ribonucleotide residues are continuous.Alternatively, the ribonucleotide residues are interspersed in betweennon-ribonucleotide residues. In some cases, the nucleic acid of areporter has only ribonucleotide residues. In some cases, the nucleicacid of a reporter has only deoxyribonucleotide residues. In some cases,the nucleic acid comprises nucleotides resistant to cleavage by theprogrammable nuclease described herein. In some cases, the nucleic acidof a reporter comprises synthetic nucleotides. In some cases, thenucleic acid of a reporter comprises at least one ribonucleotide residueand at least one non-ribonucleotide residue. In some cases, the nucleicacid of a reporter is 5-20, 5-15, 5-10, 7-20, 7-15, or 7-10 nucleotidesin length. In some cases, the nucleic acid of a reporter is from 3 to20, from 4 to 10, from 5 to 10, or from 5 to 8 nucleotides in length. Insome cases, the nucleic acid of a reporter comprises at least one uracilribonucleotide. In some cases, the nucleic acid of a reporter comprisesat least two uracil ribonucleotides. Sometimes the nucleic acid of areporter has only uracil ribonucleotides. In some cases, the nucleicacid of a reporter comprises at least one adenine ribonucleotide. Insome cases, the nucleic acid of a reporter comprises at least twoadenine ribonucleotide. In some cases, the nucleic acid of a reporterhas only adenine ribonucleotides. In some cases, the nucleic acid of areporter comprises at least one cytosine ribonucleotide. In some cases,the nucleic acid of a reporter comprises at least two cytosineribonucleotide. In some cases, the nucleic acid of a reporter comprisesat least one guanine ribonucleotide. In some cases, the nucleic acid ofa reporter comprises at least two guanine ribonucleotide. A nucleic acidof a reporter can comprise only unmodified ribonucleotides, onlyunmodified deoxyribonucleotides, or a combination thereof. In somecases, the nucleic acid of a reporter is from 5 to 12 nucleotides inlength. In some cases, the nucleic acid of a reporter is at least 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases,the nucleic acid of a reporter is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleotides in length. For cleavage by a programmable nucleasecomprising Cas13, a nucleic acid of a reporter can be 5, 8, or 10nucleotides in length. For cleavage by a programmable nucleasecomprising Cas12, a nucleic acid of a reporter can be 10 nucleotides inlength.

The single stranded nucleic acid of a reporter comprises a detectionmoiety capable of generating a first detectable signal. Sometimes thedetector nucleic acid comprises a protein capable of generating asignal. A signal can be a calorimetric, potentiometric, amperometric,optical (e.g., fluorescent, colorimetric, etc.), or piezo-electricsignal. In some cases, a detection moiety is on one side of the cleavagesite. Optionally, a quenching moiety is on the other side of thecleavage site. Sometimes the quenching moiety is a fluorescencequenching moiety. In some cases, the quenching moiety is 5′ to thecleavage site and the detection moiety is 3′ to the cleavage site. Insome cases, the detection moiety is 5′ to the cleavage site and thequenching moiety is 3′ to the cleavage site. Sometimes the quenchingmoiety is at the 5′ terminus of the nucleic acid of a reporter.Sometimes the detection moiety is at the 3′ terminus of the nucleic acidof a reporter. In some cases, the detection moiety is at the 5′ terminusof the nucleic acid of a reporter. In some cases, the quenching moietyis at the 3′ terminus of the nucleic acid of a reporter. In some cases,the single-stranded nucleic acid of a reporter is at least onepopulation of the single-stranded nucleic acid capable of generating afirst detectable signal. In some cases, the single-stranded nucleic acidof a reporter is a population of the single stranded nucleic acidcapable of generating a first detectable signal. Optionally, there ismore than one population of single-stranded nucleic acid of a reporter.In some cases, there are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,20, 30, 40, 50, or greater than 50, or any number spanned by the rangeof this list of different populations of single-stranded nucleic acidsof a reporter capable of generating a detectable signal. In some cases,there are from 2 to 50, from 3 to 40, from 4 to 30, from 5 to 20, orfrom 6 to 10 different populations of single-stranded nucleic acids of areporter capable of generating a detectable signal.

TABLE 4 Exemplary Single Stranded Nucleic Acids in a Reporter5′ Detection Moiety* Sequence (SEQ ID NO) 3′ Quencher* /56-FAM/rUrUrUrUrU (SEQ ID NO: 111) /3IABkFQ/ /5IRD700/rUrUrUrUrU (SEQ ID NO: 111) /3IRQC1N/ /5TYE665/rUrUrUrUrU (SEQ ID NO: 111) /3IAbRQSp/ /5Alex594N/rUrUrUrUrU (SEQ ID NO: 111) /3IAbRQSp/ /5ATTO633N/rUrUrUrUrU (SEQ ID NO: 111) /3IAbRQSp/ /56-FAM/rUrUrUrUrUrUrUrU(SEQ ID NO: 112) /3IABkFQ/ /5IRD700/rUrUrUrUrUrUrUrU(SEQ ID NO: 112) /3IRQC1N/ /5TYE665/rUrUrUrUrUrUrUrU(SEQ ID NO: 112) /3IAbRQSp/ /5Alex594N/rUrUrUrUrUrUrUrU(SEQ ID NO: 112) /3IAbRQSp/ /5ATTO633N/rUrUrUrUrUrUrUrU(SEQ ID NO: 112) /3IAbRQSp/ /56-FAM/rUrUrUrUrUrUrUrUrUrU(SEQ ID NO: 113) /3IABkFQ/ /5IRD700/rUrUrUrUrUrUrUrUrUrU(SEQ ID NO: 113) /3IRQC1N/ /5TYE665/rUrUrUrUrUrUrUrUrUrU(SEQ ID NO: 113) /3IAbRQSp/ /5Alex594N/rUrUrUrUrUrUrUrUrUrU(SEQ ID NO: 113) /3IAbRQSp/ /5ATTO633N/rUrUrUrUrUrUrUrUrUrU(SEQ ID NO: 113) /3IAbRQSp/ /56-FAM/TTTTrUrUTTTT(SEQ ID NO: 114) /3IABkFQ/ /5IRD700/TTTTrUrUTTTT(SEQ ID NO: 114) /3IRQC1N/ /5TYE665/TTTTrUrUTTTT(SEQ ID NO: 114) /3IAbRQSp/ /5Alex594N/TTTTrUrUTTTT(SEQ ID NO: 114) /3IAbRQSp/ /5ATTO633N/TTTTrUrUTTTT(SEQ ID NO: 114) /3IAbRQSp/ /56-FAM/TTrUrUTT(SEQ ID NO: 115) /3IABkFQ/ /5IRD700/ TTrUrUTT(SEQ ID NO: 115)/3IRQC1N/ /5TYE665/ TTrUrUTT(SEQ ID NO: 115) /3IAbRQSp/ /5Alex594N/TTrUrUTT(SEQ ID NO: 115) /3IAbRQSp/ /5ATTO633N/ TTrUrUTT(SEQ ID NO: 115)/3IAbRQSp/ /56-FAM/ TArArUGC(SEQ ID NO: 116) /3IABkFQ/ /5IRD700/TArArUGC(SEQ ID NO: 116) /3IRQC1N/ /5TYE665/ TArArUGC(SEQ ID NO: 116)/3IAbRQSp/ /5Alex594N/ TArArUGC(SEQ ID NO: 116) /3IAbRQSp/ /5ATTO633N/TArArUGC(SEQ ID NO: 116) /3IAbRQSp/ /56-FAM/ TArUrGGC(SEQ ID NO: 117)/3IABkFQ/ /5IRD700/ TArUrGGC(SEQ ID NO: 117) /3IRQC1N/ /5TYE665/TArUrGGCSEQ ID NO: 117) /3IAbRQSp/ /5Alex594N/ TArUrGGC(SEQ ID NO: 117)/3IAbRQSp/ /5ATTO633N/ TArUrGGC(SEQ ID NO: 117) /3IAbRQSp/ /56-FAM/rUrUrUrUrU(SEQ ID NO: 118) /3IABkFQ/ /5IRD700/rUrUrUrUrU(SEQ ID NO: 118) /3IRQC1N/ /5TYE665/rUrUrUrUrU(SEQ ID NO: 118) /3IAbRQSp/ /5Alex594N/rUrUrUrUrU(SEQ ID NO: 118) /3IAbRQSp/ /5ATTO633N/rUrUrUrUrU(SEQ ID NO: 118) /3IAbRQSp/ /56-FAM/ TTATTATT (SEQ ID NO: 119)/3IABkFQ/ /56-FAM/ TTATTATT (SEQ ID NO: 119) /3IABkFQ/ /5IRD700/TTATTATT (SEQ ID NO: 119) /3IRQC1N/ /5TYE665/ TTATTATT (SEQ ID NO: 119)/3IAbRQSp/ /5Alex594N/ TTATTATT (SEQ ID NO: 119) /3IAbRQSp/ /5ATTO633N/TTATTATT (SEQ ID NO: 119) /3IAbRQSp/ /56-FAM/ TTTTTT (SEQ ID NO: 120)/3IABkFQ/ /56-FAM/ TTTTTTTT (SEQ ID NO: 121) /3IABkFQ/ /56-FAM/TTTTTTTTTT (SEQ ID NO: 122) /3IABkFQ/ /56-FAM/TTTTTTTTTTTT (SEQ ID NO: 123) /3IABkFQ/ /56-FAM/TTTTTTTTTTTTTT (SEQ ID NO: 124) /3IABkFQ/ /56-FAM/AAAAAA (SEQ ID NO: 125) /3IABkFQ/ /56-FAM/ CCCCCC (SEQ ID NO: 126)/3IABkFQ/ /56-FAM/ GGGGGG (SEQ ID NO: 127) /3IABkFQ/ /56-FAM/TTATTATT (SEQ ID NO: 119) /3IABkFQ/ /56-FAM/: 5′ 6-Fluorescein(Integrated DNA Technologies) /3IABkFQ/: 3′ Iowa Black FQ (IntegratedDNA Technologies) /5IRD700/: 5′ IRDye 700 (Integrated DNA Technologies)/5TYE665/: 5′ TYE 665 (Integrated DNA Technologies) /5Alex594N/:5′ Alexa Fluor 594 (NHS Ester) (Integrated DNA Technologies)/5ATTO633N/: 5′ ATTO TM 633 (NHS Ester) (Integrated DNA Technologies)/3IRQC1N/: 3′ IRDye QC-1 Quencher (Li-Cor) /3IAbRQSp/: 3′ Iowa Black RQ(Integrated DNA Technologies) rU: uracil ribonucleotide rG: guanineribonucleotide *This Table refers to the detection moiety and quenchermoiety as their tradenames and their source is identified. However,alternatives, generics, or non-tradename moieties with similar functionfrom other sources can also be used.

A detection moiety can be an infrared fluorophore. A detection moietycan be a fluorophore that emits fluorescence in the range of from 500 nmand 720 nm. A detection moiety can be a fluorophore that emitsfluorescence in the range of from 500 nm and 720 nm. In some cases, thedetection moiety emits fluorescence at a wavelength of 700 nm or higher.In other cases, the detection moiety emits fluorescence at about 660 nmor about 670 nm. In some cases, the detection moiety emits fluorescencein the range of from 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660to 670, 670 to 680, 690 to 690, 690 to 700, 700 to 710, 710 to 720, or720 to 730 nm. In some cases, the detection moiety emits fluorescence inthe range from 450 nm to 750 nm, from 500 nm to 650 nm, or from 550 to650 nm. A detection moiety can be a fluorophore that emits a detectablefluorescence signal in the same range as 6-Fluorescein, IRDye 700, TYE665, Alex Fluor, or ATTO TM 633 (NHS Ester). A detection moiety can befluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594,or ATTO TM 633 (NHS Ester). A detection moiety can be a fluorophore thatemits a fluorescence in the same range as 6-Fluorescein (Integrated DNATechnologies), IRDye 700 (Integrated DNA Technologies), TYE 665(Integrated DNA Technologies), Alex Fluor 594 (Integrated DNATechnologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies).A detection moiety can be fluorescein amidite, 6-Fluorescein (IntegratedDNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665(Integrated DNA Technologies), Alex Fluor 594 (Integrated DNATechnologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies).Any of the detection moieties described herein can be from anycommercially available source, can be an alternative with a similarfunction, a generic, or a non-tradename of the detection moietieslisted.

A detection moiety can be chosen for use based on the type of sample tobe tested. For example, a detection moiety that is an infraredfluorophore is used with a urine sample. As another example, SEQ ID NO:111 with a fluorophore that emits a fluorescence around 520 nm is usedfor testing in non-urine samples, and SEQ ID NO: 118 with a fluorophorethat emits a fluorescence around 700 nm is used for testing in urinesamples.

A quenching moiety can be chosen based on its ability to quench thedetection moiety. A quenching moiety can be a non-fluorescentfluorescence quencher. A quenching moiety can quench a detection moietythat emits fluorescence in the range of from 500 nm and 720 nm. Aquenching moiety can quench a detection moiety that emits fluorescencein the range of from 500 nm and 720 nm. In some cases, the quenchingmoiety quenches a detection moiety that emits fluorescence at awavelength of 700 nm or higher. In other cases, the quenching moietyquenches a detection moiety that emits fluorescence at about 660 nm orabout 670 nm. In some cases, the quenching moiety quenches a detectionmoiety that emits fluorescence in the range of from 500 to 520, 500 to540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 690 to 690, 690 to700, 700 to 710, 710 to 720, or 720 to 730 nm. In some cases, thequenching moiety quenches a detection moiety that emits fluorescence inthe range from 450 nm to 750 nm, from 500 nm to 650 nm, or from 550 to650 nm. A quenching moiety can quench fluorescein amidite,6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHSEster). A quenching moiety can be Iowa Black RQ, Iowa Black FQ or IRDyeQC-1 Quencher. A quenching moiety can quench fluorescein amidite,6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNATechnologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594(Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (IntegratedDNA Technologies). A quenching moiety can be Iowa Black RQ (IntegratedDNA Technologies), Iowa Black FQ (Integrated DNA Technologies) or IRDyeQC-1 Quencher (LiCor). Any of the quenching moieties described hereincan be from any commercially available source, can be an alternativewith a similar function, a generic, or a non-tradename of the quenchingmoieties listed.

The generation of the detectable signal from the release of thedetection moiety indicates that cleavage by the programmable nucleasehas occurred and that the sample contains the target nucleic acid. Insome cases, the detection moiety comprises a fluorescent dye. Sometimesthe detection moiety comprises a fluorescence resonance energy transfer(FRET) pair. In some cases, the detection moiety comprises an infrared(IR) dye. In some cases, the detection moiety comprises an ultraviolet(UV) dye. Alternatively or in combination, the detection moietycomprises a polypeptide. Sometimes the detection moiety comprises abiotin. Sometimes the detection moiety comprises at least one of avidinor streptavidin. In some instances, the detection moiety comprises apolysaccharide, a polymer, or a nanoparticle. In some instances, thedetection moiety comprises a gold nanoparticle or a latex nanoparticle.

A detection moiety can be any moiety capable of generating acalorimetric, potentiometric, amperometric, optical (e.g., fluorescent,colorimetric, etc.), or piezo-electric signal. A nucleic acid of areporter, sometimes, is protein-nucleic acid that is capable ofgenerating a calorimetric, potentiometric, amperometric, optical (e.g.,fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavageof the nucleic acid. Often a calorimetric signal is heat produced aftercleavage of the nucleic acids of a reporter. Sometimes, a calorimetricsignal is heat absorbed after cleavage of the nucleic acids of areporter. A potentiometric signal, for example, is electrical potentialproduced after cleavage of the nucleic acids of a reporter. Anamperometric signal can be movement of electrons produced after thecleavage of nucleic acid of a reporter. Often, the signal is an opticalsignal, such as a colorimetric signal or a fluorescence signal. Anoptical signal is, for example, a light output produced after thecleavage of the nucleic acids of a reporter. Sometimes, an opticalsignal is a change in light absorbance between before and after thecleavage of nucleic acids of a reporter. Often, a piezo-electric signalis a change in mass between before and after the cleavage of the nucleicacid of a reporter.

Often, the protein-nucleic acid is an enzyme-nucleic acid. The enzymemay be sterically hindered when present as in the enzyme-nucleic acid,but then functional upon cleavage from the nucleic acid. Often, theenzyme is an enzyme that produces a reaction with a substrate. An enzymecan be invertase. Often, the substrate of invertase is sucrose. A DNSreagent produces a colorimetric change when invertase converts sucroseto glucose. In some cases, it is preferred that the nucleic acid (e.g.,DNA) and invertase are conjugated using a heterobifunctional linker viasulfo-SMCC chemistry. Sometimes the protein-nucleic acid is asubstrate-nucleic acid. Often the substrate is a substrate that producesa reaction with an enzyme.

A protein-nucleic acid may be attached to a solid support. The solidsupport, for example, is a surface. A surface can be an electrode.Sometimes the solid support is a bead. Often the bead is a magneticbead. Upon cleavage, the protein is liberated from the solid andinteracts with other mixtures. For example, the protein is an enzyme,and upon cleavage of the nucleic acid of the enzyme-nucleic acid, theenzyme flows through a chamber into a mixture comprising the substrate.When the enzyme meets the enzyme substrate, a reaction occurs, such as acolorimetric reaction, which is then detected. As another example, theprotein is an enzyme substrate, and upon cleavage of the nucleic acid ofthe enzyme substrate-nucleic acid, the enzyme flows through a chamberinto a mixture comprising the enzyme. When the enzyme substrate meetsthe enzyme, a reaction occurs, such as a calorimetric reaction, which isthen detected.

Often, the signal is a colorimetric signal or a signal visible by eye.In some instances, the signal is fluorescent, electrical, chemical,electrochemical, or magnetic. A signal can be a calorimetric,potentiometric, amperometric, optical (e.g., fluorescent, colorimetric,etc.), or piezo-electric signal. In some cases, the detectable signal isa colorimetric signal or a signal visible by eye. In some instances, thedetectable signal is fluorescent, electrical, chemical, electrochemical,or magnetic. In some cases, the first detection signal is generated bybinding of the detection moiety to the capture molecule in the detectionregion, where the first detection signal indicates that the samplecontained the target nucleic acid. Sometimes the system is capable ofdetecting more than one type of target nucleic acid, wherein the systemcomprises more than one type of guide nucleic acid and more than onetype of nucleic acid of a reporter. In some cases, the detectable signalis generated directly by the cleavage event. Alternatively or incombination, the detectable signal is generated indirectly by the signalevent. Sometimes the detectable signal is not a fluorescent signal. Insome instances, the detectable signal is a colorimetric or color-basedsignal. In some cases, the detected target nucleic acid is identifiedbased on its spatial location on the detection region of the supportmedium. In some cases, the second detectable signal is generated in aspatially distinct location than the first generated signal.

In some cases, the threshold of detection, for a subject method ofdetecting a single stranded target nucleic acid in a sample, is lessthan or equal to 10 nM. The term “threshold of detection” is used hereinto describe the minimal amount of target nucleic acid that must bepresent in a sample in order for detection to occur. For example, when athreshold of detection is 10 nM, then a signal can be detected when atarget nucleic acid is present in the sample at a concentration of 10 nMor more. In some cases, the threshold of detection is less than or equalto 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005 nM, 0.001 nM,0.0005 nM, 0.0001 nM, 0.00005 nM, 0.00001 nM, 10 pM, 1 pM, 500 fM, 250fM, 100 fM, 50 fM, 10 fM, 5 fM, 1 fM, 500 attomole (aM), 100 aM, 50 aM,10 aM, or 1 aM. In some cases, the threshold of detection is in a rangeof from 1 aM to 1 nM, 1 aM to 500 pM, 1 aM to 200 pM, 1 aM to 100 pM, 1aM to 10 pM, 1 aM to 1 pM, 1 aM to 500 fM, 1 aM to 100 fM, 1 aM to 1 fM,1 aM to 500 aM, 1 aM to 100 aM, 1 aM to 50 aM, 1 aM to 10 aM, 10 aM to 1nM, 10 aM to 500 pM, 10 aM to 200 pM, 10 aM to 100 pM, 10 aM to 10 pM,10 aM to 1 pM, 10 aM to 500 fM, 10 aM to 100 fM, 10 aM to 1 fM, 10 aM to500 aM, 10 aM to 100 aM, 10 aM to 50 aM, 100 aM to 1 nM, 100 aM to 500pM, 100 aM to 200 pM, 100 aM to 100 pM, 100 aM to 10 pM, 100 aM to 1 pM,100 aM to 500 fM, 100 aM to 100 fM, 100 aM to 1 fM, 100 aM to 500 aM,500 aM to 1 nM, 500 aM to 500 pM, 500 aM to 200 pM, 500 aM to 100 pM,500 aM to 10 pM, 500 aM to 1 pM, 500 aM to 500 fM, 500 aM to 100 fM, 500aM to 1 fM, 1 fM to 1 nM, 1 fM to 500 pM, 1 fM to 200 pM, 1 fM to 100pM, 1 fM to 10 pM, 1 fM to 1 pM, 10 fM to 1 nM, 10 fM to 500 pM, 10 fMto 200 pM, 10 fM to 100 pM, 10 fM to 10 pM, 10 fM to 1 pM, 500 fM to 1nM, 500 fM to 500 pM, 500 fM to 200 pM, 500 fM to 100 pM, 500 fM to 10pM, 500 fM to 1 pM, 800 fM to 1 nM, 800 fM to 500 pM, 800 fM to 200 pM,800 fM to 100 pM, 800 fM to 10 pM, 800 fM to 1 pM, from 1 pM to 1 nM, 1pM to 500 pM, 1 pM to 200 pM, 1 pM to 100 pM, or 1 pM to 10 pM. In somecases, the threshold of detection in a range of from 800 fM to 100 pM, 1pM to 10 pM, 10 fM to 500 fM, 10 fM to 50 fM, 50 fM to 100 fM, 100 fM to250 fM, or 250 fM to 500 fM. In some cases the threshold of detection isin a range of from 2 aM to 100 pM, from 20 aM to 50 pM, from 50 aM to 20pM, from 200 aM to 5 pM, or from 500 aM to 2 pM. In some cases, theminimum concentration at which a single stranded target nucleic acid isdetected in a sample is in a range of from 1 aM to 1 nM, 10 aM to 1 nM,100 aM to 1 nM, 500 aM to 1 nM, 1 fM to 1 nM, 1 fM to 500 pM, 1 fM to200 pM, 1 fM to 100 pM, 1 fM to 10 pM, 1 fM to 1 pM, 10 fM to 1 nM, 10fM to 500 pM, 10 fM to 200 pM, 10 fM to 100 pM, 10 fM to 10 pM, 10 fM to1 pM, 500 fM to 1 nM, 500 fM to 500 pM, 500 fM to 200 pM, 500 fM to 100pM, 500 fM to 10 pM, 500 fM to 1 pM, 800 fM to 1 nM, 800 fM to 500 pM,800 fM to 200 pM, 800 fM to 100 pM, 800 fM to 10 pM, 800 fM to 1 pM, 1pM to 1 nM, 1 pM to 500 pM, from 1 pM to 200 pM, 1 pM to 100 pM, or 1 pMto 10 pM. In some cases, the minimum concentration at which a singlestranded target nucleic acid is detected in a sample is in a range offrom 2 aM to 100 pM, from 20 aM to 50 pM, from 50 aM to 20 pM, from 200aM to 5 pM, or from 500 aM to 2 pM. In some cases, the minimumconcentration at which a single stranded target nucleic acid can bedetected in a sample is in a range of from 1 aM to 100 pM. In somecases, the minimum concentration at which a single stranded targetnucleic acid can be detected in a sample is in a range of from 1 fM to100 pM. In some cases, the minimum concentration at which a singlestranded target nucleic acid can be detected in a sample is in a rangeof from 10 fM to 100 pM. In some cases, the minimum concentration atwhich a single stranded target nucleic acid can be detected in a sampleis in a range of from 800 fM to 100 pM. In some cases, the minimumconcentration at which a single stranded target nucleic acid can bedetected in a sample is in a range of from 1 pM to 10 pM. In some cases,the devices, systems, fluidic devices, kits, and methods describedherein detect a target single-stranded nucleic acid in a samplecomprising a plurality of nucleic acids such as a plurality ofnon-target nucleic acids, where the target single-stranded nucleic acidis present at a concentration as low as 1 aM, 10 aM, 100 aM, 500 aM, 1fM, 10 fM, 500 fM, 800 fM, 1 pM, 10 pM, 100 pM, or 1 pM.

In some embodiments, the target nucleic acid is present in the cleavagereaction at a concentration of about 10 nM, about 20 nM, about 30 nM,about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about90 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 μM,about 10 μM, or about 100 μM. In some embodiments, the target nucleicacid is present in the cleavage reaction at a concentration of from 10nM to 20 nM, from 20 nM to 30 nM, from 30 nM to 40 nM, from 40 nM to 50nM, from 50 nM to 60 nM, from 60 nM to 70 nM, from 70 nM to 80 nM, from80 nM to 90 nM, from 90 nM to 100 nM, from 100 nM to 200 nM, from 200 nMto 300 nM, from 300 nM to 400 nM, from 400 nM to 500 nM, from 500 nM to600 nM, from 600 nM to 700 nM, from 700 nM to 800 nM, from 800 nM to 900nM, from 900 nM to 1 μM, from 1 μM to 10 μM, from 10 μM to 100 μM, from10 nM to 100 nM, from 10 nM to 1 μM, from 10 nM to 10 μM, from 10 nM to100 μM, from 100 nM to 1 μM, from 100 nM to 10 μM, from 100 nM to 100μM, or from 1 μM to 100 μM. In some embodiments, the target nucleic acidis present in the cleavage reaction at a concentration of from 20 nM to50 μM, from 50 nM to 20 μM, or from 200 nM to 5 μM.

In some cases, the methods, compositions, reagents, enzymes, and kitsdescribed herein may be used to detect a target single-stranded nucleicacid in a sample where the sample is contacted with the reagents for apredetermined length of time sufficient for the trans cleavage to occuror cleavage reaction to reach completion. In some cases, the devices,systems, fluidic devices, kits, and methods described herein detect atarget single-stranded nucleic acid in a sample where the sample iscontacted with the reagents for no greater than 60 minutes. Sometimesthe sample is contacted with the reagents for no greater than 120minutes, 110 minutes, 100 minutes, 90 minutes, 80 minutes, 70 minutes,60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes,30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4minutes, 3 minutes, 2 minutes, or 1 minute. Sometimes the sample iscontacted with the reagents for at least 120 minutes, 110 minutes, 100minutes, 90 minutes, 80 minutes, 70 minutes, 60 minutes, 55 minutes, 50minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20minutes, 15 minutes, 10 minutes, or 5 minutes. In some cases, the sampleis contacted with the reagents for from 5 minutes to 120 minutes, from 5minutes to 100 minutes, from 10 minutes to 90 minutes, from 15 minutesto 45 minutes, or from 20 minutes to 35 minutes. In some cases, thedevices, systems, fluidic devices, kits, and methods described hereincan detect a target nucleic acid in a sample in less than 10 hours, lessthan 9 hours, less than 8 hours, less than 7 hours, less than 6 hours,less than 5 hours, less than 4 hours, less than 3 hours, less than 2hours, less than 1 hour, less than 50 minutes, less than 45 minutes,less than 40 minutes, less than 35 minutes, less than 30 minutes, lessthan 25 minutes, less than 20 minutes, less than 15 minutes, less than10 minutes, less than 9 minutes, less than 8 minutes, less than 7minutes, less than 6 minutes, or less than 5 minutes. In some cases, thedevices, systems, fluidic devices, kits, and methods described hereincan detect a target nucleic acid in a sample in from 5 minutes to 10hours, from 10 minutes to 8 hours, from 15 minutes to 6 hours, from 20minutes to 5 hours, from 30 minutes to 2 hours, or from 45 minutes to 1hour.

When a guide nucleic acid binds to a target nucleic acid, theprogrammable nuclease's trans cleavage activity can be initiated, andnucleic acids of a reporter can be cleaved, resulting in the detectionof fluorescence. The guide nucleic acid may be a non-naturally occurringguide nucleic acid. A non-naturally occurring guide nucleic acid maycomprise an engineered sequence having a repeat and a spacer thathybridizes to a target nucleic acid sequence of interest. Anon-naturally occurring guide nucleic acid may be recombinantlyexpressed or chemically synthezised. Nucleic acid reporters can comprisea detection moiety, wherein the nucleic acid reporter can be cleaved bythe activated programmable nuclease, thereby generating a signal. Somemethods as described herein can a method of assaying for a targetnucleic acid in a sample comprises contacting the sample to a complexcomprising a guide nucleic acid comprising a segment that is reversecomplementary to a segment of the target nucleic acid and a programmablenuclease that exhibits sequence independent cleavage upon forming acomplex comprising the segment of the guide nucleic acid binding to thesegment of the target nucleic acid; and assaying for a signal indicatingcleavage of at least some protein-nucleic acids of a population ofprotein-nucleic acids, wherein the signal indicates a presence of thetarget nucleic acid in the sample and wherein absence of the signalindicates an absence of the target nucleic acid in the sample. Thecleaving of the nucleic acid of a reporter using the programmablenuclease may cleave with an efficiency of 50% as measured by a change ina signal that is calorimetric, potentiometric, amperometric, optical(e.g., fluorescent, colorimetric, etc.), or piezo-electric, asnon-limiting examples. Some methods as described herein can be a methodof detecting a target nucleic acid in a sample comprising contacting thesample comprising the target nucleic acid with a guide nucleic acidtargeting a target nucleic acid segment, a programmable nuclease capableof being activated when complexed with the guide nucleic acid and thetarget nucleic acid segment, a single stranded nucleic acid of areporter comprising a detection moiety, wherein the nucleic acid of areporter is capable of being cleaved by the activated programmablenuclease, thereby generating a first detectable signal, cleaving thesingle stranded nucleic acid of a reporter using the programmablenuclease that cleaves as measured by a change in color, and measuringthe first detectable signal on the support medium. The cleaving of thesingle stranded nucleic acid of a reporter using the programmablenuclease may cleave with an efficiency of 50% as measured by a change incolor. In some cases, the cleavage efficiency is at least 40%, 50%, 60%,70%, 80%, 90%, or 95% as measured by a change in color. The change incolor may be a detectable colorimetric signal or a signal visible byeye. The change in color may be measured as a first detectable signal.The first detectable signal can be detectable within 5 minutes ofcontacting the sample comprising the target nucleic acid with a guidenucleic acid targeting a target nucleic acid segment, a programmablenuclease capable of being activated when complexed with the guidenucleic acid and the target nucleic acid segment, and a single strandednucleic acid of a reporter comprising a detection moiety, wherein thenucleic acid of a reporter is capable of being cleaved by the activatednuclease. The first detectable signal can be detectable within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, or 120 minutes of contacting the sample.In some embodiments, the first detectable signal can be detectablewithin from 1 to 120, from 5 to 100, from 10 to 90, from 15 to 80, from20 to 60, or from 30 to 45 minutes of contacting the sample.

In some cases, the methods, reagents, enzymes, and kits described hereindetect a target single-stranded nucleic acid with a programmablenuclease and a single-stranded nucleic acid of a reporter in a samplewhere the sample is contacted with the reagents for a predeterminedlength of time sufficient for trans cleavage of the single strandednucleic acid of a reporter. In a preferred embodiment, a Cas13aprogrammable nuclease us used to detect the presence of asingle-stranded DNA target nucleic acid. For example, a programmablenuclease is LbuCas13a that detects a target nucleic acid and a singlestranded nucleic acid of a reporter comprises two adjacent uracilnucleotides with a green detectable moiety that is detected uponcleavage. As another example, a programmable nuclease is LbaCas13a thatdetects a target nucleic acid and a single-stranded nucleic acid of areporter comprises two adjacent adenine nucleotides with a reddetectable moiety that is detected upon cleavage.

Buffers

The reagents described herein can also include buffers, which arecompatible with the methods, compositions, reagents, enzymes, and kitsdisclosed herein. Buffers can be referred to herein as a “highperformance buffer” or an “activity buffer” and are compatible withdifferent programmable nucleases described herein. Compositionsincluding the high performance buffers and programmable nucleasesdescribed herein exhibit superior and efficient transcollateral cleavageactivity in the various methods described herein (e.g., DETECTR assaymethods for assaying for a target nucleic acid). Any of the methods,compositions, reagents, enzymes, or kits disclosed herein may comprise abuffer (e.g., a high performance buffer or an activity buffer). Thesebuffers are compatible with the other reagents, samples, and supportmediums as described herein for detection of an ailment, such as adisease, cancer, or genetic disorder, or genetic information, such asfor phenotyping, genotyping, or determining ancestry. A buffer, asdescribed herein, can enhance the assay detection a target nucleic acid,such as enhancing a method of assaying for a target nucleic acid in asample, comprises: contacting the sample to a complex comprising a guidenucleic acid comprising a segment that is reverse complementary to asegment of the target nucleic acid and a programmable nuclease thatexhibits sequence independent cleavage upon forming a complex comprisingthe segment of the guide nucleic acid binding to the segment of thetarget nucleic acid, wherein the sample comprises at least one nucleicacid comprising at least 50% sequence identity to the segment of thetarget nucleic acid; and assaying for cleavage of at least one detectornucleic acids of a population of detector nucleic acids, wherein thecleavage indicates a presence of the target nucleic acid in the sampleand wherein absence of the cleavage indicates an absence of the targetnucleic acid in the sample. The buffer can increase the discriminationof the programmable nuclease of the segment of the target nucleic acidand the at least one nucleic acid comprising at least 50% sequenceidentity to the segment of the target nucleic acid. For example, thebuffer increases the discrimination between the segment of the targetnucleic acid comprising a single nucleotide mutation and the at leastone nucleic acid comprising a variant of the single nucleotide mutationof the segment of the target nucleic acid. Sometimes, the bufferincreases the discrimination between the segment of the target nucleicacid comprising deletion and the at least one nucleic acid comprising avariant of the segment of the target nucleic acid. The methods asdescribed herein can be performed in the buffer.

In some embodiments, a buffer may comprise one or more of a bufferingagent, a salt, a crowding agent, or a detergent, or any combinationthereof. A buffer may comprise a reducing agent. A buffer may comprise acompetitor. Exemplary buffering agent include HEPES, Tris, andimidazole. A buffer may comprise HEPES, Tris, or any combinationthereof. A buffer compatible with a programmable nuclease (e.g., SEQ IDNO: 11) may comprise HEPES. A buffer may comprise HEPES, Tris, or anycombination thereof. A buffer compatible with a programmable nuclease(e.g., SEQ ID NO: 1) may comprise Tris. In some embodiments, a buffercompatible with a Cas12 programmable nuclease (e.g., SEQ ID NO: 11)comprises a buffering agent at a concentration of from 10 mM to 40 mM.In some embodiments, a buffer compatible with a Cas12 programmablenuclease (e.g., SEQ ID NO: 11) comprises a buffering agent at aconcentration of about 20 mM. A buffer compatible with a programmablenuclease may comprise a buffering agent at a concentration of from 5 mMto 100 mM. A buffer compatible with a programmable nuclease may comprisea buffering agent at a concentration of from 10 mM to 30 mM. Acomposition (e.g., a composition comprising a programmable nuclease) mayhave a pH of from 7 to 8. A buffer compatible with a programmablenuclease (e.g., SEQ ID NO: 1 or SEQ ID NO: 11) may comprise a bufferingagent at a concentration of from 1 mM to 50 mM. A buffer compatible witha programmable nuclease (e.g., SEQ ID NO: 1 or SEQ ID NO: 11) maycomprise a buffering agent at a concentration of from 1 mM to 30 mM. Abuffer compatible with a programmable nuclease (e.g., SEQ ID NO: 1 orSEQ ID NO: 11) may comprise a buffering agent at a concentration ofabout 20 mM.

Exemplary salts include NaCl, KCl, magnesium acetate, potassium acetate,and MgCl₂. A buffer may comprise potassium acetate, magnesium acetate,sodium chloride, magnesium chloride, or any combination thereof. In someembodiments, a buffer compatible with a Cas12 programmable nuclease(e.g., SEQ ID NO: 11) comprises a salt at a concentration of from 5 mMto 100 mM. In some embodiments, a buffer compatible with a Cas12programmable nuclease (e.g., SEQ ID NO: 11) comprises a salt at aconcentration of from 5 mM to 10 mM. A buffer compatible with aprogrammable nuclease may comprise a salt at a concentration of from 5mM to 100 mM. A buffer compatible with a programmable nuclease maycomprise a salt at a concentration of from 5 mM to 10 mM. In someembodiments, a buffer compatible with a programmable nuclease (e.g., SEQID NO: 11 or SEQ ID NO: 104) comprises a salt from 1 mM to 60 mM. Insome embodiments, a buffer compatible with a programmable nuclease(e.g., SEQ ID NO: 11) comprises a salt from 1 mM to 10 mM. In someembodiments, a buffer compatible with a programmable nuclease (e.g., SEQID NO: 1) comprises a salt at about 105 mM. In some embodiments, abuffer compatible with a programmable nuclease (e.g., SEQ ID NO: 104)comprises a salt at about 55 mM. In some embodiments, a buffercompatible with a programmable nuclease (e.g., SEQ ID NO: 11) comprisesa salt at about 7 mM. In some embodiments, a buffer compatible with aprogrammable nuclease (e.g., SEQ ID NO: 11) comprises a salt, whereinthe salt comprises potassium acetate and magnesium acetate. In someembodiments, a buffer compatible with a programmable nuclease (e.g., SEQID NO: 1) comprises a salt, wherein the salt comprises sodium chlorideand magnesium chloride. In some embodiments, a buffer compatible with aprogrammable nuclease (e.g., SEQ ID NO: 104) comprises a salt, whereinthe salt comprises potassium chloride and magnesium chloride.

Exemplary crowding agents include glycerol and bovine serum albumin. Abuffer may comprise glycerol. A crowding agent may reduce the volume ofsolvent available for other molecules in the solution, therebyincreasing the effective concentrations of said molecules. In someembodiments, a buffer compatible with a Cas12 programmable nuclease(e.g., SEQ ID NO: 11) comprises a crowding agent at a concentration offrom 0.5% (v/v) to 2% (v/v). In some embodiments, a buffer compatiblewith a Cas12 programmable nuclease (e.g., SEQ ID NO: 11) comprises acrowding agent at a concentration of about 1% (v/v). A buffer compatiblewith a programmable nuclease may comprise a crowding agent at aconcentration of from 1% (v/v) to 5% (v/v). A buffer compatible with aprogrammable nuclease may comprise a crowding agent at a concentrationof from 0.5% (v/v) to 10% (v/v).

Exemplary detergents include Tween, Triton-X, and IGEPAL. A buffer maycomprise Tween, Triton-X, or any combination thereof. A buffercompatible with a programmable nuclease (e.g., SEQ ID NO: 11) maycomprise Triton-X. A buffer compatible with a programmable nuclease(e.g., SEQ ID NO: 104) may comprise IGEPAL CA-630. In some embodiments,a buffer compatible with a programmable nuclease (e.g., SEQ ID NO: 11)comprises a detergent at a concentration of 2% (v/v) or less. In someembodiments, a buffer compatible with a programmable nuclease (e.g., SEQID NO: 11) comprises a detergent at a concentration of about 0.00016%(v/v). A buffer compatible with a programmable nuclease may comprise adetergent at a concentration of 2% (v/v) or less. A buffer compatiblewith a programmable nuclease (e.g., SEQ ID NO: 11 or SEQ ID NO: 104) maycomprise a detergent at a concentration of from 0.00001% (v/v) to 0.01%(v/v). A buffer compatible with a programmable nuclease (e.g., SEQ IDNO: 104) may comprise a detergent at a concentration of about 0.01%(v/v).

Exemplary reducing agents comprise dithiothreitol (DTT),8-mercaptoethanol (BME), or tris(2-carboxyethyl)phosphine (TCEP). Abuffer may comprise DTT. A buffer compatible with a programmablenuclease (e.g., SEQ ID NO: 1) may comprise DTT. A buffer compatible witha programmable nuclease may comprise a reducing agent at a concentrationof from 0.01 mM to 100 mM. A buffer compatible with a programmablenuclease may comprise a reducing agent at a concentration of from 0.1 mMto 10 mM. A buffer compatible with a programmable nuclease may comprisea reducing agent at a concentration of from 0.5 mM to 2 mM. A buffercompatible with a programmable nuclease (e.g., SEQ ID NO: 1) maycomprise a reducing agent at a concentration of from 0.01 mM to 100 mM.A buffer compatible with a programmable nuclease (e.g., SEQ ID NO: 1)may comprise a reducing agent at a concentration of from 0.1 mM to 10mM. A buffer compatible with a programmable nuclease (e.g., SEQ IDNO: 1) may comprise a reducing agent at a concentration of about 1 mM.

A buffer compatible with a programmable nuclease may comprise acompetitor. Exemplary competitors compete with the target nucleic acidor the detector nucleic acid for cleavage by the programmable nuclease.Exemplary competitors include heparin, and imidazole, and salmon spermDNA. A buffer may comprise heparin. A buffer compatible with aprogrammable nuclease may comprise a competitor at a concentration offrom 1 μg/mL to 100 μg/mL. A buffer compatible with a programmablenuclease may comprise a competitor at a concentration of from 40 μg/mLto 60 μg/mL.

In some embodiments, a buffer compatible with a programmable nuclease(e.g., SEQ ID NO: 1, SEQ ID NO: 11, or SEQ ID NO: 104) comprises acrowding agent or a competitor. For example, the crowding agent ispresent from 1% (v/v) to 10% (v/v). In some embodiments, the crowdingagent or competitor is present from 1% (v/v) to 5% (v/v). In someembodiments, a buffer compatible with a programmable nuclease (e.g., SEQID NO: 1, or SEQ ID NO: 104) comprises a crowding agent or competitorpresent at about to 5% (v/v). In some embodiments, a buffer compatiblewith a programmable nuclease (e.g., SEQ ID NO: 11) comprises a crowdingagent or competitor present at about 1% (v/v). In some embodiments, abuffer compatible with a programmable nuclease (e.g., SEQ ID NO: 1)comprises a crowding agent or competitor present from 1 μg/mL to 100μg/mL. In some embodiments, a buffer compatible with a programmablenuclease (e.g., SEQ ID NO: 1) comprises a crowding agent or competitorpresent from 30 μg/mL to 70 μg/mL. In some embodiments, a buffercompatible with a programmable nuclease (e.g., SEQ ID NO: 1) comprises acrowding agent or competitor present at about 50 μg/mL. In someembodiments, a buffer compatible with a programmable nuclease (e.g., SEQID NO: 104) comprises a crowding agent or competitor present from 1 mMto 30 mM. In some embodiments, a buffer compatible with a programmablenuclease (e.g., SEQ ID NO: 104) comprises a crowding agent or competitorpresent from 1 mM to 50 mM. In some embodiments, a buffer compatiblewith a programmable nuclease (e.g., SEQ ID NO: 104) comprises a crowdingagent or competitor present at about 20 mM. The crowding agent orcompetitor may be selected from the group consisting of: glycerol,heparin, bovine serum albumin, imidazole, and any combination thereof.In some embodiments, a buffer compatible with a programmable nuclease(e.g., SEQ ID NO: 11) comprises glycerol. In some embodiments, a buffercompatible with a programmable nuclease (e.g., SEQ ID NO: 1) comprisesglycerol and heparin. In some embodiments, a buffer compatible with aprogrammable nuclease (e.g., SEQ ID NO: 104) comprises glycerol, BSA,and imidazole.

Sometimes, a method used herein comprises: contacting a programmablenuclease comprising a polypeptide having endonuclease activity and aguide nucleic acid to a target nucleic acid in a buffer comprisingheparin. The heparin is present, for example, at a concentration of from1 to 100 μg/ml heparin. Often, the heparin is present at a concentrationof from 40 to 60 μg/ml heparin. Sometimes, the heparin is present at aconcentration 50 μg/ml heparin. Often, the buffer comprises NaCl. TheNaCl is present, for example, at a concentration of from 1 to 200 mMNaCl. Sometimes, the NaCl is present at a concentration of from 80 to120 mM NaCl. Often, the NaCl is present at a concentration of 100 mMNaCl.

In some embodiments, the buffer comprises heparin. The buffer cancomprise 50 μg/ml heparin. Sometimes, the buffer comprises 5 μg/ml, 10μg/ml, 15 μg/ml, 20 μg/ml, 25 μg/ml, 30 μg/ml, 35 μg/ml, 40 μg/ml, 45μg/ml, 50 μg/ml, 55 μg/ml, 60 μg/ml, 65 μg/ml, 70 μg/ml, 75 μg/ml, 80μg/ml, 85 μg/ml, 90 μg/ml, 95 μg/ml, or 100 μg/ml of heparin, or anyvalue within these values. Often, the buffer comprises from 40 μg/ml to60 μg/ml heparin. Often, a buffer may comprise from 40 μg/ml to 60 μg/mlheparin. Preferably, a specificity buffer may comprise 50 μg/ml heparin.Preferrably, a high sensitivity buffer may not contain heparin.

In some embodiments, the buffer comprises NaCl. The buffer can comprise100 mM NaCl. Sometimes, the buffer comprises 50 mM, 55 mM, 60 mM, 65 mM,70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115mM, 120 mM, 125 mM, 130 mM, 140 mM, 150 mM, or 200 mM of NaCl, or anyvalue within these values. Often, the buffer comprises from 75 mM to 125mM NaCl. Preferrably, a high specificity buffer may comprise 100 mMNaCl. Preferrably, a high sensitivity buffer may not contain NaCl.

In some embodiments, the buffer comprises heparin and NaCl. The buffercan comprise 50 μg/ml heparin and 100 mM NaCl. Sometimes, the buffercomprises 5 μg/ml, 10 μg/ml, 15 μg/ml, 20 μg/ml, 25 μg/ml, 30 μg/ml, 35μg/ml, 40 μg/ml, 45 μg/ml, 50 μg/ml, 55 μg/ml, 60 μg/ml, 65 μg/ml, 70μg/ml, 75 μg/ml, 80 μg/ml, 85 μg/ml, 90 μg/ml, 95 μg/ml, or 100 μg/ml ofheparin, or any value within these values, and 50 mM, 55 mM, 60 mM, 65mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM,115 mM, 120 mM, 125 mM, 130 mM, 140 mM, 150 mM, or 200 mM of NaCl, orany value within these values. Preferrably, a high specificity buffermay comprise 100 mM NaCl and 50 μg/ml heparin. Preferrably, a highsensitivity buffer may not contain NaCl and may not contain heparin.

A high specificity buffer can comprise 20 mM Tris pH 8.0, 100 mM NaCl, 5mM MgCl₂, 1 mm DTT, 5% (v/v) glycerol, and 50 μg/ml heparin.

In contrast a high sensitivity buffer comprises the high specificitybuffer as described above, but without the heparin and NaCl (e.g., 20 mMTris pH 8.0, 5 mM MgCl₂, 1 mm DTT, 5% (v/v) glycerol).

Sometimes, a method used herein comprises: contacting a programmablenuclease comprising a polypeptide having endonuclease activity and aguide nucleic acid to a target nucleic acid in a buffer comprisingheparin. The heparin is present, for example, at a concentration of from1 to 100 μg/ml heparin. Often, the heparin is present at a concentrationof from 40 to 60 μg/ml heparin. Sometimes, the heparin is present at aconcentration 50 μg/ml heparin. Often, the buffer comprises NaCl. TheNaCl is present, for example, at a concentration of from 1 to 200 mMNaCl. Sometimes, the NaCl is present at a concentration of from 80 to120 mM NaCl. Often, the NaCl is present at a concentration of 100 mMNaCl.

As described herein, nucleic acid sequences comprising DNA may bedetected using a DNA-activated programmable RNA nuclease and otherreagents disclosed herein. Additionally, detection by a DNA-activatedprogrammable RNA nuclease, which can cleave RNA reporters, allows formultiplexing with other programmable nucleases, such as a DNA-activatedprogrammable DNA nuclease that can cleave DNA reporters (e.g., Type VCRISPR enzyme). The methods described herein can also include the use ofbuffers, which are compatible with the methods disclosed herein. Forexample, a buffer comprises 20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl₂,and 5% (v/v) glycerol. In some instances the buffer comprises from 0 to100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10.0 to 5, 5 to 10.5 to15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4,15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM HEPES pH 6.8. Thebuffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200,0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5,5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM KCl.Preferrably, a buffer may comprise 25 to 75 mM KCl. More preferably, abuffer may comprise 50 mM KCl. In other instances the buffer comprises 0to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM MgCl₂.Preferrably, a buffer may comprise 1 to 10 mM MgCl₂. More preferably, abuffer may comprise 5 mM MgCl₂. The buffer can comprise 0 to 25, 0 to20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30% (v/v)glycerol. The buffer can comprise 0 to 30, 2 to 25, or 10 to 20% (v/v)glycerol. Preferrably, the buffer may comprise 0% (v/v) to 10% (v/v)glycerol. More preferably, a buffer may comprise 5% (v/v) glycerol. Inan preferred example, a buffer may comprise 50 mM KCl, 5 mM MgCl₂, and5% (v/v) glycerol.

In some embodiments, a buffer compatible with a Cas12 programmablenuclease (e.g., SEQ ID NO: 11) comprises a HEPES buffering agent. Insome embodiments, a buffer compatible with a Cas12 programmable nuclease(e.g., SEQ ID NO: 11) comprises a salt, wherein the salt comprisespotassium acetate, magnesium acetate, sodium chloride, magnesiumchloride, potassium chloride, or any combination thereof. In someembodiments, a buffer compatible with a Cas12 programmable nuclease(e.g., SEQ ID NO: 11) comprises a glycerol crowding agent. In someembodiments, a buffer compatible with a Cas12 programmable nuclease(e.g., SEQ ID NO: 11) comprises a detergent, wherein the detergent isTween, Triton-X, or any combination thereof. In some embodiments, abuffer compatible with a Cas12 programmable nuclease (e.g., SEQ ID NO:11) comprises a pH of from 7 to 8. In some embodiments, a buffercompatible with a programmable nuclease (e.g., SEQ ID NO: 11 or SEQ IDNO: 104) comprises a pH of about 7.5. In some embodiments, a buffercompatible with a programmable nuclease (e.g., SEQ ID NO: 1) comprises apH of about 8.

In some embodiments, a buffer compatible with a programmable nucleasemay comprise a salt at less than about 110 mM and wherein the buffercomprises a pH of from 7 to 8. In some embodiments, the salt is from 1mM to 110 mM. In some embodiments, a buffer compatible with a Cas12programmable nuclease (e.g., SEQ ID NO: 11) comprises a pH of about 7.5.In some embodiments, a buffer (e.g., a buffer comprising about 20 mMHEPES, about 2 mM potassium acetate, about 5 mM magnesium acetate, about1% (v/v) glycerol, about 0.00016% (v/v) Triton-X, and a pH of about 7.5)is compatible with a programmable nuclease comprising at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 95%, at least 97%, at least 99%, or 100% sequence identity to SEQID NO: 11. In some embodiments, a buffer (e.g., a buffer comprisingabout 20 mM Tris, about 100 mM sodium chloride, about 5 mM magnesiumchloride, about 5% (v/v) glycerol, about 50 ug/mL heparin, about 1 mMDTT, and a pH of about 8) is compatible with a programmable nucleasecomprising at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 92%, at least 95%, at least 97%, at least 99%, or100% sequence identity to SEQ ID NO: 1. In some embodiments, a buffer(e.g., a buffer comprising about 50 mM potassium chloride, about 5 mMmagnesium chloride, about 10 ug/ml bovine serum albumin, about 5% (v/v)glycerol, about 20 mM imidazole, about 0.01% (v/v) IGEPAL CA-630, and apH of about 7.5) is compatible with a programmable nuclease comprisingat least 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 92%, at least 95%, at least 97%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 104. Any of the buffers or compositions describedherein may comprise a guide nucleic acid (e.g., a non-naturallyoccurring guide nucleic acid). Any of the buffers or compositionsdescribed herein may comprise a detector nucleic acid.

As another example, a buffer comprises 100 mM Imidazole pH 7.5; 250 mMKCl, 25 mM MgCl₂, 50 μg/mL BSA, 0.05% (v/v) Igepal Ca-630, and 25% (v/v)Glycerol. In some instances the buffer comprises 0 to 500, 0 to 400, 0to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30,5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to300, or 150 to 250 mM Imidazole pH 7.5. In some instances, the buffercomprises 100 to 250, 100 to 200, or 150 to 200 mM Imidazole pH 7.5.Preferrably, the buffer may comprise 20 mM Imidazole pH 7.5. The buffercan comprise 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150,0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10,5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300,100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM KCl. Preferrably, abuffer may comprise 25 to 75 mM KCl. More preferably, a buffer maycomprise 50 mM KCl. In other instances the buffer comprises 0 to 100, 0to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM MgCl₂. Preferrably, abuffer may comprise 1 to 10 mM MgCl₂. More preferably, a buffer maycomprise 5 mM MgCl₂. The buffer, in some instances, comprises 0 to 100,0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 50, 5 to 75, 5to 100, 10 to 20, 10 to 50, 10 to 75, 10 to 100, 25 to 50, 25 to 75 25to 100, 50 to 75, or 50 to 100 μg/mL BSA. In some instances, the buffercomprises 0 to 1, 0 to 0.5, 0 to 0.25, 0 to 0.01, 0 to 0.05, 0 to 0.025,0 to 0.01, 0.01 to 0.025, 0.01 to 0.05, 0.01 to 0.1, 0.01 to 0.25, 0.01,to 0.5, 0.01 to 1, 0.025 to 0.05, 0.025 to 0.1, 0.025, to 0.5, 0.025 to1, 0.05 to 0.1, 0.05 to 0.25, 0.05 to 0.5, 0.05 to 0.75, 0.05 to 1, 0.1to 0.25, 0.1 to 0.5, or 0.1 to 1% (v/v) Igepal Ca-630. The buffer cancomprise 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5to 25, 5 to 30% (v/v) glycerol. The buffer can comprise 0 to 30, 2 to25, or 10 to 20% (v/v) glycerol. Preferrably, the buffer may comprise 0%(v/v) to 10% (v/v) glycerol. More preferably, a buffer may comprise 5%(v/v) glycerol. While reagent (e.g., crowding agents or detergents)concentrations may be described in terms of percent volume per volume(v/v), a percent may also indicate percent weight per volume (w/v).

Stability

Present in this disclosure are stable compositions of the reagents andthe programmable nuclease system for use in the methods as discussedherein. The reagents and programmable nuclease system described hereinmay be stable in various storage conditions including refrigerated,ambient, and accelerated conditions. Disclosed herein are stablereagents. The stability may be measured for the reagents andprogrammable nuclease system themselves or the reagents and programmablenuclease system present on the support medium.

In some instances, stable as used herein refers to a reagents havingabout 5% w/w or less total impurities at the end of a given storageperiod. Stability may be assessed by HPLC or any other known testingmethod. The stable reagents may have about 10% w/w, about 5% w/w, about4% w/w, about 3% w/w, about 2% w/w, about 1% w/w, or about 0.5% w/wtotal impurities at the end of a given storage period. The stablereagents may have from 0.5% w/w to 10% w/w, from 1% w/w to 8% w/w, from2% w/w to 7% w/w, or from 3% w/w to 5% w/w total impurities at the endof a given storage period.

In some embodiments, stable as used herein refers to a reagents andprogrammable nuclease system having about 10% or less loss of detectionactivity at the end of a given storage period and at a given storagecondition. Detection activity can be assessed by known positive sampleusing a known method. Alternatively or combination, detection activitycan be assessed by the sensitivity, accuracy, or specificity. In someembodiments, the stable reagents has about 10%, about 9%, about 8%,about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, orabout 0.5% loss of detection activity at the end of a given storageperiod. In some embodiments, the stable reagents has from 0.5% to 10%,from 1% to 8%, from 2% to 7%, or from 3% to 5% loss of detectionactivity at the end of a given storage period.

In some embodiments, the stable composition has zero loss of detectionactivity at the end of a given storage period and at a given storagecondition. The given storage condition may comprise humidity of equal toor less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%relative humidity. The controlled storage environment may comprisehumidity from 0% to 50% relative humidity, from 0% to 40% relativehumidity, from 0% to 30% relative humidity, from 0% to 20% relativehumidity, or from 0% to 10% relative humidity. The controlled storageenvironment may comprise humidity from 10% to 80%, from 10% to 70%, from10% to 60%, from 20% to 50%, from 20% to 40%, or from 20% to 30%relative humidity. The controlled storage environment may comprisetemperatures of about −100° C., about −80° C., about −20° C., about 4°C., about 25° C. (room temperature), or about 40° C. The controlledstorage environment may comprise temperatures from −80° C. to 25° C., orfrom −100° C. to 40° C. The controlled storage environment may comprisetemperatures from −20° C. to 40° C., from −20° C. to 4° C., or from 4°C. to 40° C. The controlled storage environment may protect the systemor kit from light or from mechanical damage. The controlled storageenvironment may be sterile or aseptic or maintain the sterility of thelight conduit. The controlled storage environment may be aseptic orsterile.

A kit of this disclosure can be packaged to be stored for extendedperiods of time prior to use. The kit or system may be packaged to avoiddegradation of the kit or system. The packaging may include desiccantsor other agents to control the humidity within the packaging. Thepackaging may protect the kit or system from mechanical damage orthermal damage. The packaging may protect the kit or system fromcontamination of the reagents and programmable nuclease system. The kitor system may be transported under conditions similar to the storageconditions that result in high stability of the reagent or little lossof reagent activity. The packaging may be configured to provide andmaintain sterility of the kit. The kit can be compatible with standardmanufacturing and shipping operations.

Multiplexing

The compositions and methods disclosed herein can be carried out formultiplexed detection. The compositions and methods for multiplexeddetection are compatible with the DETECTR assay methods disclosedherein. The compositions and methods for multiplexed detection describedhere are compatible with any of the programmable nucleases disclosedherein (e.g., a programmable nuclease with at least 60% sequenceidentity to SEQ ID NO: 11) and use of said programmable nuclease in amethod of detecting a target nucleic acid. The compositions and methodsfor multiplexed detection described here are compatible with any of thecompositions comprising a programmable nuclease and a buffer, which hasbeen developed to improve the function of the programmable nuclease(e.g., a programmable nuclease and a buffer with low salt (about 110 mMor less) and a pH of 7 to 8) and use of said compositions in a method ofdetecting a target nucleic acid. The compositions and methods formultiplexed detection described here are compatible with any of themethods disclosed herein including methods of assaying for at least onebase difference (e.g., assaying for a SNP or a base mutation) in atarget nucleic acid sequence, methods of assaying for a target nucleicacid that lacks a PAM by amplifying the target nucleic acid sequence tointroduce a PAM, and compositions used in introducing a PAM viaamplification into the target nucleic acid sequence. These methods ofmultiplexing are, for example, consistent with fluidic devices fordetection of a target nucleic acid sequence within the sample, whereinthe fluidic device may comprise multiple pumps, valves, reservoirs, andchambers for sample preparation, amplification of a target nucleic acidsequence within the sample, mixing with a programmable nuclease, anddetection of a detectable signal arising from cleavage of the nucleicacids of a reporter by the programmable nuclease within the fluidicsystem itself.

The methods described herein can be multiplexed in a number of ways.These methods of multiplexing are, for example, consistent with theassay methods disclosed herein for detection of a target nucleic acidwithin the sample when the target nucleic acid, such as multiplexing amethod of assaying for a target nucleic acid in a sample, comprises:contacting the sample to a complex comprising a guide nucleic acidcomprising a segment that is reverse complementary to a segment of thetarget nucleic acid and a programmable nuclease that exhibits sequenceindependent cleavage upon forming a complex comprising the segment ofthe guide nucleic acid binding to the segment of the target nucleicacid, wherein the sample comprises at least one nucleic acid comprisingat least 50% sequence identity to the segment of the target nucleicacid; and assaying for cleavage of at least one detector nucleic acidsof a population of detector nucleic acids, wherein the cleavageindicates a presence of the target nucleic acid in the sample andwherein absence of the cleavage indicates an absence of the targetnucleic acid in the sample. The guide nucleic acid may be anon-naturally occurring guide nucleic acid. A non-naturally occurringguide nucleic acid may comprise an engineered sequence having a repeatand a spacer that hybridizes to a target nucleic acid sequence ofinterest. A non-naturally occurring guide nucleic acid may berecombinantly expressed or chemically synthezised.

In some embodiments, the target nucleic acid for multiplexed detectionlacks a PAM. A method of multiplexed assaying for a target nucleic acidsegment in a sample, wherein the target nucleic acid segment lacks a PAMsequence, may comprise amplifying the target nucleic acid segment usinga primer having a region that is reverse complementary to the targetnucleic acid segment and a region that has a PAM sequence reversecomplement, thereby generating a PAM target nucleic acid having a PAMsequence adjacent to target sequence of an amplification product;contacting the PAM target nucleic acid to PAM-dependent sequencespecific nuclease complex comprising a guide nucleic acid and aprogrammable nuclease that exhibits sequence independent cleavage uponforming a complex comprising the segment of the guide nucleic acidbinding to the segment of the PAM target nucleic acid; and assaying forcleavage of at least one detector nucleic acid of a population ofdetector nucleic acids, wherein the cleavage indicates a presence of thetarget nucleic acid in the sample and wherein the absence of thecleavage indicates an absence of the target nucleic acid in the sample.

Methods consistent with the present disclosure include a multiplexingmethod of assaying for a target nucleic acid in a sample. A multiplexingmethod comprises contacting the sample to a complex comprising a guidenucleic acid comprising a segment that is reverse complementary to asegment of the target nucleic acid (e.g., DNA) and a programmablenuclease (e.g., a DNA-activated programmable RNA nuclease, such asCas13) that exhibits sequence-independent cleavage upon forming acomplex comprising the segment of the guide nucleic acid binding to thetarget nucleic acid; and assaying for a signal indicating cleavage of atleast some protein-nucleic acids of a population of protein-nucleicacids, wherein the signal indicates a presence of the target nucleicacid in the sample and wherein absence of the signal indicates anabsence of the target nucleic acid in the sample. As another example,multiplexing method of assaying for a target nucleic acid in a sample,for example, comprises: a) contacting the sample to a complex comprisinga guide nucleic acid comprising a segment that is reverse complementaryto a segment of the target nucleic acid and a programmable nuclease thatexhibits sequence independent cleavage upon forming a complex comprisingthe segment of the guide nucleic acid binding to the segment of thetarget nucleic acid; b) contacting the complex to a substrate; c)contacting the substrate to a reagent that differentially reacts with acleaved substrate; and d) assaying for a signal indicating cleavage ofthe substrate, wherein the signal indicates a presence of the targetnucleic acid in the sample and wherein absence of the signal indicatesan absence of the target nucleic acid in the sample. Often, thesubstrate is an enzyme-nucleic acid. Sometimes, the substrate is anenzyme substrate-nucleic acid.

Multiplexing can be either spatial multiplexing wherein multipledifferent target nucleic acids at the same time, but the reactions arespatially separated. Often, the multiple target nucleic acids aredetected using the same programmable nuclease, but different guidenucleic acids. The multiple target nucleic acids sometimes are detectedusing the different programmable nucleases. For example, a DNA-activatedprogrammable RNA nuclease and a DNA-activated programmable DNA nucleasecan both be used in a single assay to directly detect DNA targetsencoding different sequences. The activated DNA-activated programmableRNA nuclease cleaves an RNA reporter, generating a first detectablesignal and the activated DNA-activated programmable DNA nuclease cleavesa DNA reporter, generating a second detectable signal. In someembodiments, the first and second detectable signals are different, andthose allow simultaneous detection of more than one target DNA sequencesusing two programmable nucleases. In some embodiments, the DNA-activatedprogrammable DNA nuclease and the DNA-activated programmable RNAnuclease are complexed to a guide nucleic acid that hybridizes to thesame target DNA. The activated DNA-activated programmable RNA nucleasecleaves an RNA reporter, generating a first detectable signal and theDNA-activated programmable DNA nuclease cleaves a DNA reporter,generating a second detectable signal. The first detectable signal andthe second detectable signal can be the same, thus, allowing for signalamplification by cleavage of reporters by two different programmablenucleases that are activated by the same target DNA.

Sometimes, multiplexing can be single reaction multiplexing whereinmultiple different target nucleic acids are detected in a singlereaction volume. Often, at least two different programmable nucleasesare used in single reaction multiplexing. For example, multiplexing canbe enabled by immobilization of multiple categories of nucleic acids ofa reporter within a fluidic system, to enable detection of multipletarget nucleic acids within a single fluidic system. Multiplexing allowsfor detection of multiple target nucleic acids in one kit or system. Insome cases, the multiple target nucleic acids comprise different targetnucleic acids to a virus, a bacterium, or a pathogen responsible for onedisease. In some cases, the multiple target nucleic acids comprisedifferent target nucleic acids associated with a cancer or geneticdisorder. Multiplexing for one disease, cancer, or genetic disorderincreases at least one of sensitivity, specificity, or accuracy of theassay to detect the presence of the disease in the sample. In somecases, the multiple target nucleic acids comprise target nucleic acidsdirected to different viruses, bacteria, or pathogens responsible formore than one disease. In some cases, multiplexing allows fordiscrimination between multiple target nucleic acids, such as targetnucleic acids that comprise different genotypes of the same bacteria orpathogen responsible for a disease, for example, for a wild-typegenotype of a bacteria or pathogen and for genotype of a bacteria orpathogen comprising a mutation, such as a single nucleotide polymorphism(SNP) or deletion that can confer resistance to a treatment, such asantibiotic treatment. For example, multiplexing comprises method ofassaying comprising a single assay for a microorganism species using afirst programmable nuclease and an antibiotic resistance pattern in amicroorganism using a second programmable nuclease. Sometimes,multiplexing allows for discrimination between multiple target nucleicacids of different HPV strains, for example, HPV16 and HPV18. In somecases, the multiple target nucleic acids comprise target nucleic acidsdirected to different cancers or genetic disorders. Often, multiplexingallows for discrimination between multiple target nucleic acids, such astarget nucleic acids that comprise different genotypes, for example, fora wild-type genotype and for SNP genotype. Multiplexing for multiplediseases, cancers, or genetic disorders provides the capability to testa panel of diseases from a single sample. For example, multiplexing formultiple diseases can be valuable in a broad panel testing of a newpatient or in epidemiological surveys. Often multiplexing is used foridentifying bacterial pathogens in sepsis or other diseases associatedwith multiple pathogens.

Furthermore, signals from multiplexing can be quantified. For example, amethod of quantification for a disease panel comprises assaying for aplurality of unique target nucleic acids in a plurality of aliquots froma sample, assaying for a control nucleic acid control in a secondaliquot of the sample, and quantifying a plurality of signals of theplurality of unique target nucleic acids by measuring signals producedby cleavage of nucleic acids of a reporter compared to the signalproduced in the second aliquot. Often the plurality of unique targetnucleic acids are from a plurality of bacterial pathogens in the sample.Sometimes the quantification of a signal of the plurality correlateswith a concentration of a unique target nucleic acid of the pluralityfor the unique target nucleic acid of the plurality that produced thesignal of the plurality. The disease panel can be for any communicabledisease, such as sepsis.

The methods described herein can be multiplexed by variousconfigurations of the reagents and the support medium. In some cases,the kit or system is designed to have multiple support mediums encasedin a single housing. Sometimes, the multiple support mediums housed in asingle housing share a single sample pad. The single sample pad may beconnected to the support mediums in various designs such as a branchingor a radial formation. Alternatively, each of the multiple supportmediums has its own sample pad. In some cases, the kit or system isdesigned to have a single support medium encased in a housing, where thesupport medium comprises multiple detection spots for detecting multipletarget nucleic acids. Sometimes, the reagents for multiplexed assayscomprise multiple guide nucleic acids, multiple programmable nucleases,and multiple single stranded detector nucleic acids, where a combinationof one of the guide nucleic acids, one of the programmable nucleases,and one of the single stranded detector nucleic acids detects one targetnucleic acid and can provide a detection spot on the detection region.In some cases, the combination of a guide nucleic acid, a programmablenuclease, and a single stranded detector nucleic acid configured todetect one target nucleic acid is mixed with at least one othercombination in a single reagent chamber. In some cases, the combinationof a guide nucleic acid, a programmable nuclease, and a single strandeddetector nucleic acid configured to detect one target nucleic acid ismixed with at least one other combination on a single support medium.When these combinations of reagents are contacted with the sample, thereaction for the multiple target nucleic acids occurs simultaneously inthe same medium or reagent chamber. Sometimes, this reacted sample isapplied to the multiplexed support medium described herein.

In some cases, the combination of a guide nucleic acid, a programmablenuclease, and a single stranded detector nucleic acid configured todetect one target nucleic acid is provided in its own reagent chamber orits own support medium. In this case, multiple reagent chambers orsupport mediums are provided in the device, kit, or system, where onereagent chamber is designed to detect one target nucleic acid. In thiscase, multiple support mediums are used to detect the panel of diseases,cancers, or genetic disorders of interest.

In some instances, multiplexed detection detects at least 2 differenttarget nucleic acids in a single reaction. In some instances,multiplexed detection detects at least 3 different target nucleic acidsin a single reaction. In some instances, multiplexed detection detectsat least 4 different target nucleic acids in a single reaction. In someinstances, multiplexed detection detects at least 5 different targetnucleic acids in a single reaction. In some cases, multiplexed detectiondetects at least 6, 7, 8, 9, or 10 different target nucleic acids in asingle reaction. In some instances, the multiplexed kits detect at least2 different target nucleic acids in a single kit. In some instances, themultiplexed kits detect at least 3 different target nucleic acids in asingle kit. In some instances, the multiplexed kits detect at least 4different target nucleic acids in a single kit. In some instances, themultiplexed kits detect at least 5 different target nucleic acids in asingle kit. In some instances, the multiplexed kits detect at least 6,7, 8, 9, or 10 different target nucleic acids in a single kit. In someinstances, the multiplexed kits detect from 2 to 10, from 3 to 9, from 4to 8, or from 5 to 7 different target nucleic acids in a single kit.

Detection Methods

Disclosed herein are methods of assaying for a target nucleic acid asdescribed herein wherein a signal is detected. The methods of assayingfor a target nucleic acid wherein a signal is detected are compatiblewith the DETECTR assay methods disclosed herein. The methods of assayingfor a target nucleic acid wherein a signal is detected, as describedherein, are compatible with any of the programmable nucleases disclosedherein (e.g., a programmable nuclease with at least 60% sequenceidentity to SEQ ID NO: 11) and use of said programmable nuclease in amethod of detecting a target nucleic acid. The methods of assaying for atarget nucleic acid wherein a signal is detected, as described herein,are compatible with any of the compositions comprising a programmablenuclease and a buffer, which has been developed to improve the functionof the programmable nuclease (e.g., a programmable nuclease and a bufferwith low salt (about 110 mM or less) and a pH of 7 to 8) and use of saidcompositions in a method of detecting a target nucleic acid. The methodsof assaying for a target nucleic acid wherein a signal is detected, asdescribed herein, are compatible with any of the methods disclosedherein including methods of assaying for at least one base difference(e.g., assaying for a SNP or a base mutation) in a target nucleic acidsequence, methods of assaying for a target nucleic acid that lacks a PAMby amplifying the target nucleic acid sequence to introduce a PAM, andcompositions used in introducing a PAM via amplification into the targetnucleic acid sequence. A method of assaying for a segment of a targetnucleic acid in a sample may comprise contacting the sample to adetector nucleic acid any of the compositions described herein (e.g., acomposition comprising a programmable nuclease of SEQ ID NO: 11),wherein the guide nucleic acid hybridizes to a segment of the targetnucleic acid, and assaying for a signal produced by cleavage of thedetector nucleic acid. In some embodiments, the programmable nuclease(e.g., a Cas12 programmable nuclease) cleaves the detector nucleic acidupon hybridization of the guide nucleic acid to the segment of thetarget nucleic acid. In some embodiments, the signal produced bycleavage of the detector nucleic acid may be at least two-fold greaterwhen the segment of the target nucleic acid is present in the samplethan the signal when the sample lacks the segment of the target nucleicacid and wherein the subject has a disease when the segment of thetarget nucleic acid is present.

In some embodiments, the methods disclosed herein are methods ofassaying for a target deoxyribonucleic acid as described herein using aDNA-activated programmable RNA nuclease wherein a signal is detected.For example, a method of assaying for a target nucleic acid in a samplecomprises contacting the sample to a complex comprising a guide nucleicacid comprising a segment that is reverse complementary to a segment ofthe target nucleic acid and a programmable nuclease that exhibitssequence independent cleavage upon forming a complex comprising thesegment of the guide nucleic acid binding to the segment of the targetnucleic acid; and assaying for a signal indicating cleavage of at leastsome protein-nucleic acids of a population of protein-nucleic acids,wherein the signal indicates a presence of the target nucleic acid inthe sample and wherein absence of the signal indicates an absence of thetarget nucleic acid in the sample. In some embodiments, the samplecomprises at least one nucleic acid comprising at least 50% sequenceidentity to a segment of the target nucleic acid. As another example, amethod of assaying for a target nucleic acid in a sample, for example,comprises: a) contacting the sample to a complex comprising a guidenucleic acid comprising a segment that is reverse complementary to asegment of the target nucleic acid and a DNA-activated programmable RNAnuclease that exhibits sequence independent cleavage upon forming acomplex comprising the segment of the guide nucleic acid binding to thesegment of the target nucleic acid; b) contacting the complex to asubstrate; c) contacting the substrate to a reagent that differentiallyreacts with a cleaved substrate; and d) assaying for a signal indicatingcleavage of the substrate, wherein the signal indicates a presence ofthe target nucleic acid in the sample and wherein absence of the signalindicates an absence of the target nucleic acid in the sample. Often,the substrate is an enzyme-nucleic acid. Sometimes, the substrate is anenzyme substrate-nucleic acid. As described herein, nucleic acidsequences comprising DNA may be detected using a DNA-activatedprogrammable RNA nuclease and other reagents disclosed herein.

In some embodiments, a method of assaying for a target nucleic acid in asample comprises a sample, wherein the target nucleic acid segment lacksa PAM. For example, a method of assaying for a target nucleic acidsegment in a sample, wherein the target nucleic acid segment lacks a PAMsequence, comprises amplifying the target nucleic acid segment using aprimer having a region that is reverse complementary to the targetnucleic acid segment and a region that has a PAM sequence reversecomplement, thereby generating a PAM target nucleic acid having a PAMsequence adjacent to target sequence of an amplification product;contacting the PAM target nucleic acid to PAM-dependent sequencespecific nuclease complex comprising a guide nucleic acid and aprogrammable nuclease that exhibits sequence independent cleavage uponforming a complex comprising the segment of the guide nucleic acidbinding to the segment of the PAM target nucleic acid; and assaying forcleavage of at least one detector nucleic acid of a population ofdetector nucleic acids, wherein the cleavage indicates a presence of thetarget nucleic acid in the sample and wherein the absence of thecleavage indicates an absence of the target nucleic acid in the sample.A PAM-dependent sequence specific nuclease, often, is a programmablenuclease. Sometimes, a PAM-dependent sequence specific nuclease is aPAM-dependent sequence specific endonuclease.

Present in this disclosure are methods of assaying for a target nucleicacid as described herein. In some embodiments, the method is a method ofassaying for a target deoxyribonucleic acid using a DNA-activatedprogrammable RNA nuclease, wherein assaying comprises detecting cleavageof an RNA reporter. For example, a method of assaying for a targetnucleic acid in a sample comprises contacting the sample to a complexcomprising a guide nucleic acid comprising a segment that is reversecomplementary to a segment of the target nucleic acid and a programmablenuclease (e.g., a DNA-activated programmable RNA nuclease) that exhibitssequence independent cleavage upon forming a complex comprising thesegment of the guide nucleic acid binding to the segment of the targetnucleic acid (e.g. target deoxyribonucleic acid); and assaying for asignal indicating cleavage of at least some protein-nucleic acids of apopulation of protein-nucleic acids, wherein the signal indicates apresence of the target nucleic acid in the sample and wherein absence ofthe signal indicates an absence of the target nucleic acid in thesample. As another example, a method of assaying for a target nucleicacid in a sample, for example, comprises: a) contacting the sample to acomplex comprising a guide nucleic acid comprising a segment that isreverse complementary to a segment of the target nucleic acid and aprogrammable nuclease that exhibits sequence independent cleavage uponforming a complex comprising the segment of the guide nucleic acidbinding to the segment of the target nucleic acid; b) contacting thecomplex to a substrate; c) contacting the substrate to a reagent thatdifferentially reacts with a cleaved substrate; and d) assaying for asignal indicating cleavage of the substrate, wherein the signalindicates a presence of the target nucleic acid in the sample andwherein absence of the signal indicates an absence of the target nucleicacid in the sample. Often, the substrate is an enzyme-nucleic acid.Sometimes, the substrate is an enzyme substrate-nucleic acid.

A programmable nuclease can comprise a programmable nuclease capable ofbeing activated when complexed with a guide nucleic acid and targetnucleic acid. The programmable nuclease can become activated afterbinding of a guide nucleic acid with a target nucleic acid, in which theactivated programmable nuclease can cleave the target nucleic acid andcan have trans cleavage activity. Trans cleavage activity can benon-specific cleavage of nearby nucleic acids by the activatedprogrammable nuclease, such as trans cleavage of detector nucleic acidswith a detection moiety. Once the detector nucleic acid is cleaved bythe activated programmable nuclease, the detection moiety can bereleased from the detector nucleic acid and can generate a signal. Thesignal can be immobilized on a support medium for detection. The signalcan be visualized to assess whether a target nucleic acid comprises amodification.

Often, the signal is a colorimetric signal or a signal visible by eye.In some instances, the signal is fluorescent, electrical, chemical,electrochemical, or magnetic. In some cases, the signal is generated bybinding of the detection moiety to the capture molecule in the detectionregion, where the signal indicates that the sample contained the targetnucleic acid. Sometimes the system is capable of detecting more than onetype of target nucleic acid, wherein the system comprises more than onetype of guide nucleic acid and more than one type of detector nucleicacid. In some cases, the signal is generated directly by the cleavageevent. Alternatively, or in combination, the signal is generatedindirectly by the signal event. Sometimes the signal is not afluorescent signal. In some instances, the signal is a colorimetric orcolor-based signal. In some cases, the detected target nucleic acid isidentified based on its spatial location on the detection region of thesupport medium. In some cases, the second detectable signal is generatedin a spatially distinct location than the first generated signal.

In some cases, the threshold of detection, for a method of assaying of atarget nucleic acid described herein in a sample, is less than or equalto 10 nM. The term “threshold of detection” is used herein to describethe minimal amount of target nucleic acid that must be present in asample in order for detection to occur. For example, when a threshold ofdetection is 10 nM, then a signal can be detected when a target nucleicacid is present in the sample at a concentration of 10 nM or more. Insome cases, the threshold of detection is less than or equal to 5 nM, 1nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005 nM, 0.001 nM, 0.0005 nM,0.0001 nM, 0.00005 nM, 0.00001 nM, 10 pM, 1 pM, 500 fM, 250 fM, 100 fM,50 fM, 10 fM, 5 fM, 1 fM, 500 attomole (aM), 100 aM, 50 aM, 10 aM, or 1aM. In some cases, the threshold of detection is in a range of from 1 aMto 1 nM, 1 aM to 500 pM, 1 aM to 200 pM, 1 aM to 100 pM, 1 aM to 10 pM,1 aM to 1 pM, 1 aM to 500 fM, 1 aM to 100 fM, 1 aM to 1 fM, 1 aM to 500aM, 1 aM to 100 aM, 1 aM to 50 aM, 1 aM to 10 aM, 10 aM to 1 nM, 10 aMto 500 pM, 10 aM to 200 pM, 10 aM to 100 pM, 10 aM to 10 pM, 10 aM to 1pM, 10 aM to 500 fM, 10 aM to 100 fM, 10 aM to 1 fM, 10 aM to 500 aM, 10aM to 100 aM, 10 aM to 50 aM, 100 aM to 1 nM, 100 aM to 500 pM, 100 aMto 200 pM, 100 aM to 100 pM, 100 aM to 10 pM, 100 aM to 1 pM, 100 aM to500 fM, 100 aM to 100 fM, 100 aM to 1 fM, 100 aM to 500 aM, 500 aM to 1nM, 500 aM to 500 pM, 500 aM to 200 pM, 500 aM to 100 pM, 500 aM to 10pM, 500 aM to 1 pM, 500 aM to 500 fM, 500 aM to 100 fM, 500 aM to 1 fM,1 fM to 1 nM, 1 fM to 500 pM, 1 fM to 200 pM, 1 fM to 100 pM, 1 fM to 10pM, 1 fM to 1 pM, 10 fM to 1 nM, 10 fM to 500 pM, 10 fM to 200 pM, 10 fMto 100 pM, 10 fM to 10 pM, 10 fM to 1 pM, 500 fM to 1 nM, 500 fM to 500pM, 500 fM to 200 pM, 500 fM to 100 pM, 500 fM to 10 pM, 500 fM to 1 pM,800 fM to 1 nM, 800 fM to 500 pM, 800 fM to 200 pM, 800 fM to 100 pM,800 fM to 10 pM, 800 fM to 1 pM, from 1 pM to 1 nM, 1 pM to 500 pM, 1 pMto 200 pM, 1 pM to 100 pM, or 1 pM to 10 pM. In some cases, thethreshold of detection in a range of from 800 fM to 100 pM, 1 pM to 10pM, 10 fM to 500 fM, 10 fM to 50 fM, 50 fM to 100 fM, 100 fM to 250 fM,or 250 fM to 500 fM. In some cases, the minimum concentration at which atarget nucleic acid is detected in a sample is in a range of from 1 aMto 1 nM, 10 aM to 1 nM, 100 aM to 1 nM, 500 aM to 1 nM, 1 fM to 1 nM, 1fM to 500 pM, 1 fM to 200 pM, 1 fM to 100 pM, 1 fM to 10 pM, 1 fM to 1pM, 10 fM to 1 nM, 10 fM to 500 pM, 10 fM to 200 pM, 10 fM to 100 pM, 10fM to 10 pM, 10 fM to 1 pM, 500 fM to 1 nM, 500 fM to 500 pM, 500 fM to200 pM, 500 fM to 100 pM, 500 fM to 10 pM, 500 fM to 1 pM, 800 fM to 1nM, 800 fM to 500 pM, 800 fM to 200 pM, 800 fM to 100 pM, 800 fM to 10pM, 800 fM to 1 pM, 1 pM to 1 nM, 1 pM to 500 pM, from 1 pM to 200 pM, 1pM to 100 pM, or 1 pM to 10 pM. In some cases, the minimum concentrationat which a target nucleic acid can be detected in a sample is in a rangeof from 1 aM to 100 pM. In some cases, the minimum concentration atwhich a target nucleic acid can be detected in a sample is in a range offrom 1 fM to 100 pM. In some cases, the minimum concentration at which atarget nucleic acid can be detected in a sample is in a range of from 10fM to 100 pM. In some cases, the minimum concentration at which a targetnucleic acid can be detected in a sample is in a range of from 800 fM to100 pM. In some cases, the minimum concentration at which a targetnucleic acid can be detected in a sample is in a range of from 1 pM to10 pM. In some cases, methods described herein detect a target nucleicacid in a sample comprising a plurality of nucleic acids such as aplurality of non-target nucleic acids, where the nucleic acid is presentat a concentration as low as 1 aM, 10 aM, 100 aM, 500 aM, 1 fM, 10 fM,500 fM, 800 fM, 1 pM, 10 pM, 100 pM, or 1 pM.

In some cases, the methods described herein detect a target nucleic acidin a sample where the sample is contacted with the reagents for apredetermined length of time sufficient for the trans cleavage to occuror cleavage reaction to reach completion. In some cases, the methodsdescribed herein detect a target nucleic acid in a sample where thesample is contacted with the reagents for no greater than 60 minutes.Sometimes the sample is contacted with the reagents for no greater than120 minutes, 110 minutes, 100 minutes, 90 minutes, 80 minutes, 70minutes, 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute. Sometimes thesample is contacted with the reagents for at least 120 minutes, 110minutes, 100 minutes, 90 minutes, 80 minutes, 70 minutes, 60 minutes, 55minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes.

Some methods as described herein can be a method of assaying for atarget nucleic acid in a sample, comprises: contacting the sample to acomplex comprising a guide nucleic acid comprising a segment that isreverse complementary to a segment of the target nucleic acid and aprogrammable nuclease that exhibits sequence independent cleavage uponforming a complex comprising the segment of the guide nucleic acidbinding to the segment of the target nucleic acid, wherein the samplecomprises at least one nucleic acid comprising at least 50% sequenceidentity to the segment of the target nucleic acid; and assaying forcleavage of at least one detector nucleic acids of a population ofdetector nucleic acids, wherein the cleavage indicates a presence of thetarget nucleic acid in the sample and wherein absence of the cleavageindicates an absence of the target nucleic acid in the sample. Somemethods as described herein can be a method of assaying for a targetnucleic acid in a sample comprising: producing a PAM target nucleic acidcomprising a sequence encoding a PAM by amplifying the target nucleicacid of the sample using primers comprising the encoding the PAM;contacting the PAM target nucleic acid to a complex comprising a guidenucleic acid comprising a segment that is reverse complementary to asegment of the PAM target nucleic acid and a programmable nuclease thatexhibits sequence independent cleavage upon forming a complex comprisingthe segment of the guide nucleic acid binding to the segment of the PAMtarget nucleic acid; and assaying for a signal indicating cleavage of atleast some detector nucleic acids of a population of detector nucleicacids, wherein the signal indicates a presence of the target nucleicacid in the sample and wherein the absence of the signal indicates anabsence of the target nucleic acid in the sample. The cleaving of thedetector nucleic acid using the programmable nuclease may cleave with anefficiency of 50% as measured by a change in a signal that iscalorimetric, potentiometric, amperometric, optical (e.g., fluorescent,colorimetric, etc.), or piezo-electric, as non-limiting examples. Insome cases, the cleavage efficiency is at least 40%, 50%, 60%, 70%, 80%,90%, or 95% as measured by a change in a signal that is calorimetric,potentiometric, amperometric, optical (e.g., fluorescent, colorimetric,etc.), or piezo-electric, as non-limiting examples. The change in colormay be a detectable colorimetric signal or a signal visible by eye. Thesignal can be detectable within 5 minutes of contacting the samplecomprising the target nucleic acid to a guide nucleic acid complexedwith programmable nuclease and a detector nucleic acid comprising adetection moiety, wherein the nucleic acid of the detector nucleic iscleaved by the activated nuclease. The signal can be detectable within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 70, 80, 90, 100, 110, or 120 minutes of contacting thesample.

The methods described herein can also include the use of buffers, whichare compatible with the methods disclosed herein. For example, a buffercomprises 20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl₂, and 5% glycerol. Insome instances the buffer comprises from 0 to 100, 0 to 75, 0 to 50, 0to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40,10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20to 30, 20 to 40, or 20 to 50 mM HEPES pH 6.8. The buffer can comprise to0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200,100 to 250, 100 to 300, or 150 to 250 mM KCl. In other instances thebuffer comprises 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10,0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to50 mM MgCl₂. The buffer can comprise 0 to 25, 0 to 20, 0 to 10, 0 to 5,5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30% glycerol.

As another example, a buffer comprises 100 mM Imidazole pH 7.5; 250 mMKCl, 25 mM MgCl₂, 50 μg/mL BSA, 0.05% Igepal Ca-630, and 25% Glycerol.In some instances the buffer comprises 0 to 500, 0 to 400, 0 to 300, 0to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40,5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to250 mM Imidazole pH 7.5. The buffer can comprise to 0 to 500, 0 to 400,0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30,5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to300, or 150 to 250 mM KCl. In other instances the buffer comprises 0 to100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4,15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM MgCl₂. Thebuffer, in some instances, comprises 0 to 100, 0 to 75, 0 to 50, 0 to25, 0 to 20, 0 to 10, 0 to 5, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10to 50, 10 to 75, 10 to 100, 25 to 50, 25 to 75 25 to 100, 50 to 75, or50 to 100 μg/mL BSA. In some instances, the buffer comprises 0 to 1, 0to 0.5, 0 to 0.25, 0 to 0.01, 0 to 0.05, 0 to 0.025, 0 to 0.01, 0.01 to0.025, 0.01 to 0.05, 0.01 to 0.1, 0.01 to 0.25, 0.01, to 0.5, 0.01 to 1,0.025 to 0.05, 0.025 to 0.1, 0.025, to 0.5, 0.025 to 1, 0.05 to 0.1,0.05 to 0.25, 0.05 to 0.5, 0.05 to 0.75, 0.05 to 1, 0.1 to 0.25, 0.1 to0.5, or 0.1 to 1% Igepal Ca-630. The buffer can comprise 0 to 25, 0 to20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30%glycerol.

The methods for detection of a target nucleic acid described hereinfurther can comprises reagents protease treatment of the sample. Thesample can be treated with protease, such as Protease K, beforeamplification or before assaying for a detectable signal. Often, aprotease treatment is for no more than 15 minutes. Sometimes, theprotease treatment is for no more than 1, 5, 10, 15, 20, 25, 30, or moreminutes, or any value from 1 to 30 minutes. Sometimes, the proteasetreatment is from 1 to 30, from 5 to 25, from 10 to 20, or from 10 to 15minutes. Sometimes, the total time for the performing the methoddescribed herein is no greater than 3 hours, 2 hours, 1 hour, 50minutes, 40 minutes, 30 minutes, 20 minutes, or any value from 3 hoursto 20 minutes. Often, a method of nucleic acid detection from a rawsample comprises protease treating the sample for no more than 15minutes, amplifying (can also be referred to as pre-amplifying) thesample for no more than 15 minutes, subjecting the sample to aprogrammable nuclease-mediated detection, and assaying nuclease mediateddetection. The total time for performing this method, sometimes, is nogreater than 3 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30minutes, 20 minutes, or any value from 3 hours to 20 minutes. Often, theprotease treatment is Protease K. Often the amplifying is thermalcycling amplification. Sometimes the amplifying is isothermalamplification.

Enrichment of the Target Nucleic Acid Using a Targeting Protein

Enriching for the target nucleic acid in methods described herein canalso enhance the assay detection of the target nucleic acid, such as fora method of assaying for a target nucleic acid in a sample, comprises:contacting the sample to a complex comprising a guide nucleic acidcomprising a segment that is reverse complementary to a segment of thetarget nucleic acid and a programmable nuclease that exhibits sequenceindependent cleavage upon forming a complex comprising the segment ofthe guide nucleic acid binding to the segment of the target nucleicacid, wherein the sample comprises at least one nucleic acid comprisingat least 50% sequence identity to the segment of the target nucleicacid; and assaying for cleavage of at least one detector nucleic acidsof a population of detector nucleic acids, wherein the cleavageindicates a presence of the target nucleic acid in the sample andwherein absence of the cleavage indicates an absence of the targetnucleic acid in the sample. Often, the segment of the target nucleicacid of the methods described herein comprise a mutation and the nucleicacid comprising at least 50% sequence identity to the segment of thetarget nucleic acid comprise a variant of the mutation. In someembodiments, a target nucleic acid is enriched in a sample prior to orconcurrent with detection of the target nucleic acid using any of themethods disclosed herein.

The compositions for enrichment of target nucleic acids and methods ofuse thereof, as described herein, are compatible with the DETECTR assaymethods disclosed herein. The methods of assaying for a target nucleicacid wherein a signal is detected, as described herein, are compatiblewith any of the programmable nucleases disclosed herein (e.g., aprogrammable nuclease with at least 60% sequence identity to SEQ ID NO:11) and use of said programmable nuclease in a method of detecting atarget nucleic acid. The compositions for enrichment of target nucleicacids and methods of use thereof, as described herein, are compatiblewith any of the compositions comprising a programmable nuclease and abuffer, which has been developed to improve the function of theprogrammable nuclease (e.g., a programmable nuclease and a buffer withlow salt (about 110 mM or less) and a pH of 7 to 8) and use of saidcompositions in a method of detecting a target nucleic acid. Thecompositions for enrichment of target nucleic acids and methods of usethereof, as described herein, are compatible with any of the methodsdisclosed herein including methods of assaying for at least one basedifference (e.g., assaying for a SNP or a base mutation) in a targetnucleic acid sequence, methods of assaying for a target nucleic acidthat lacks a PAM by amplifying the target nucleic acid sequence tointroduce a PAM, and compositions used in introducing a PAM viaamplification into the target nucleic acid sequence.

A segment of the target nucleic acid may be enriched, for example, bydepleting other nucleic acid species that do not correspond to thetarget nucleic acid from the sample. A segment of the target nucleicacid may be enriched, for example, by increasing the concentration ofthe target nucleic acid in the sample. In some cases, a nucleic acidspecies that does not correspond to the target nucleic acid may be anucleic acid comprising a mutation relative to the target nucleic acid.In some cases, a nucleic acid species that does not correspond to thetarget nucleic acid may be a nucleic acid comprising a variationrelative to the target nucleic acid. In some cases, the nucleic acidspecies that does not correspond to the target nucleic acid may be aregion of a genome that does not comprise the target nucleic acid. Insome cases, a nucleic acid species that does not correspond to thetarget nucleic acid may be a nucleic acid contaminant. The segment ofthe target nucleic acid in the sample may be enriched by targeting thenucleic acid species that does not correspond to the target nucleic acidwith a protein that does not bind the segment of the target nucleicacid. For example, the protein may bind the nucleic acid comprising amutation relative to the target nucleic acid but not to the segment ofthe target nucleic acid. Targeting the nucleic acid species that doesnot correspond to the target nucleic acid with the protein that does notbind the segment of the target nucleic acid may allow for the removal ofthe targeted nucleic acid. The segment of the target nucleic acid in thesample may be enriched by targeting the target nucleic acid with aprotein that specifically binds the segment of the target nucleic acid.For example, the protein may bind the segment of target nucleic acid butnot to a nucleic acid comprising a mutation relative to the targetnucleic acid. Targeting the segment of the target nucleic acid with theprotein that specifically binds the segment of the target nucleic acidmay allow for the removal of the nucleic acids that are not targeted bythe protein or isolation of the nucleic acids targeted by the protein. Aprotein may be targeted to the segment of the target nucleic acid, orthe protein may be targeted to a nucleic acid that does not correspondto the target nucleic acid, or any combination thereof, before thecontacting of the methods described herein.

For enrichment of the segment of the target nucleic acid by targetingthe nucleic acids comprising a variant or a mutation relative to thetarget nucleic acid with a protein, the protein can be an antibody thatbinds to the variant or the mutation of the nucleic acid. Often, theprotein is a programmable nuclease without endonuclease activity.Sometimes, the protein is attached to a surface and the sample is passedthrough the protein attached to surface. The nucleic acids comprisingthe variant mutation are therefore removed from the flow through,leaving a sample with enriched target nucleic acid.

Alternatively, for enrichment of the segment of the target nucleic acidby targeting the segment of the target nucleic acid with a protein, theprotein can be an antibody that binds to the target nucleic acid. Often,the protein is a programmable nuclease without endonuclease activity.Sometimes, the protein is attached to a surface and the sample is passedthrough the protein attached to surface. The target nucleic acidstherefore bound to the protein and other nucleic acids are separatedfrom the target nucleic acids in the flow through. The bound targetnucleic acids can then be released from the protein, leaving a samplewith the enriched segments of the target nucleic acids.

Detection of a Mutation in a Target Nucleic Acid

Disclosed herein are methods of assaying for a target nucleic acid asdescribed herein that can be used for detection of a single nucleotidemutation (single nucleotide polymorphism, SNP) in a target nucleic acid.The compositions for detection of a mutation in a target nucleic acidand methods of use thereof, as described herein, are compatible with theDETECTR assay methods disclosed herein. The compositions for detectionof a mutation in a target nucleic acid and methods of use thereof, asdescribed herein, are compatible with any of the programmable nucleasesdisclosed herein (e.g., a programmable nuclease with at least 60%sequence identity to SEQ ID NO: 11) and use of said programmablenuclease in a method of detecting a target nucleic acid. Thecompositions for detection of a mutation in a target nucleic acid andmethods of use thereof, as described herein, are compatible with any ofthe compositions comprising a programmable nuclease and a buffer, whichhas been developed to improve the function of the programmable nuclease(e.g., a programmable nuclease and a buffer with low salt (about 110 mMor less) and a pH of 7 to 8) and use of said compositions in a method ofdetecting a target nucleic acid. The compositions for detection of amutation in a target nucleic acid and methods of use thereof, asdescribed herein, are compatible with any of the methods disclosedherein including methods of assaying for at least one base difference(e.g., assaying for a SNP or a base mutation) in a target nucleic acidsequence, methods of assaying for a target nucleic acid that lacks a PAMby amplifying the target nucleic acid sequence to introduce a PAM, andcompositions used in introducing a PAM via amplification into the targetnucleic acid sequence. The SNP can be a synonymous substitution or anonsynonymous substitution. The nonsynonymous substitution can be amissense substitution or a nonsense point mutation. The synonymoussubstitution can be a silent substitution. Sometimes, the methods can beused for detection of a deletion in a target nucleic acid. For example,A method of assaying for a target nucleic acid in a sample, comprises:contacting the sample to a complex comprising a guide nucleic acidcomprising a segment that is reverse complementary to a segment of thetarget nucleic acid and a programmable nuclease that exhibits sequenceindependent cleavage upon forming a complex comprising the segment ofthe guide nucleic acid binding to the segment of the target nucleicacid, wherein the sample comprises at least one nucleic acid comprisingat least 50% sequence identity to the segment of the target nucleicacid; and assaying for cleavage of at least one detector nucleic acidsof a population of detector nucleic acids, wherein the cleavageindicates a presence of the target nucleic acid in the sample andwherein absence of the cleavage indicates an absence of the targetnucleic acid in the sample. Sometimes, the target nucleic acid comprisesa mutation. Often, the mutation is a single nucleotide mutation.Alternatively, the mutation is a deletion.

A target nucleic acid may be present in a heterogenous sample, forexample a sample comprising the target nucleic acid and a nucleic acidwith less than 100% sequence identity to the target nucleic acid (e.g.,a target nucleic acid comprising a mutation and a nucleic acid that doesnot comprise the mutation). The target nucleic acid may be present inthe heterogenous sample at a minor allele frequency of 10% or less. Forexample, the target nucleic acid may comprise less than 10% of thenucleic acid population comprising the target nucleic acid comprising amutation and a nucleic acid that does not comprise the mutation. In someembodiments, the target nucleic acid may be present in the sample at aminor allele frequency of from 0.1% to 10%. In some embodiments, thetarget nucleic acid may be present in the sample at a minor allelefrequency of from 0.1% to 5%. In some embodiments, the target nucleicacid may be present in the sample at a minor allele frequency of from0.1% to 1%. In some embodiments, the segment of the nucleic acid or thesegment of the target nucleic acid comprises at least one base mutationcompared to at least one other segment of a nucleic acid in the sample.In some embodiments, the at least one base mutation is no more than 13nucleotides 3′ of the PAM in the nucleic acid or the PAM target nucleicacid. In some embodiments, the at least one base mutation is no morethan 10 nucleotides 3′ of the PAM in the nucleic acid or the PAM targetnucleic acid. In some embodiments, the at least one base mutation is nomore than 9 nucleotides 3′ of the PAM in the nucleic acid or in the PAMtarget nucleic acid. In some embodiments, the at least one base mutationis no more than 8 nucleotides 3′ of the PAM in the nucleic acid or inthe PAM target nucleic acid. In some embodiments, the at least one basemutation is a single nucleotide polymorphism.

Also disclosed herein are methods of assaying for a target nucleic acidas described herein that can be used for detection of a singlenucleotide mutation in a target nucleic acid. For example, a method ofassaying for a target nucleic acid segment in a sample, wherein thetarget nucleic acid segment lacks a PAM sequence, comprises amplifyingthe target nucleic acid segment using a primer having a region that isreverse complementary to the target nucleic acid segment and a regionthat has a PAM sequence reverse complement, thereby generating a PAMtarget nucleic acid having a PAM sequence adjacent to target sequence ofan amplification product; contacting the PAM target nucleic acid toPAM-dependent sequence specific nuclease complex comprising a guidenucleic acid and a programmable nuclease that exhibits sequenceindependent cleavage upon forming a complex comprising the segment ofthe guide nucleic acid binding to the segment of the PAM target nucleicacid; and assaying for cleavage of at least one detector nucleic acid ofa population of detector nucleic acids, wherein the cleavage indicates apresence of the target nucleic acid in the sample and wherein theabsence of the cleavage indicates an absence of the target nucleic acidin the sample. Sometimes, the target nucleic acid comprises a mutation.Often, the mutation is a single nucleotide mutation.

Methods described herein can be used to identify a mutation in a targetnucleic acid. The methods can be used to identify a single nucleotidemutation of a target nucleic acid that affects the expression of a gene.A mutation that affects the expression of gene can be a singlenucleotide mutation of a target nucleic acid within the gene, a singlenucleotide mutation of a target nucleic acid comprising RNA associatedwith the expression of a gene, or a target nucleic acid comprising asingle nucleotide mutation of a nucleic acid associated with regulationof expression of a gene, such as an RNA or a promoter, enhancer, orrepressor of the gene. A mutation that affects the expression of a genecan be a deletion of one or more nucleic acids within the gene, adeletion of one or more target nucleic acids comprising RNA associatedwith the expression of a gene, or a target nucleic acid comprising adeletion of one or more nucleic acids associated with regulation ofexpression of a gene, such as an RNA or a promoter, enhancer, orrepressor of the gene. Often, a status of a mutation is used to diagnoseor identify diseases associated with the mutation of target nucleicacid. Detection of target nucleic acids having a mutation are applicableto a number of fields, such as clinically, as a diagnostic, inlaboratories as a research tool, and in agricultural applications.Often, the mutation is a single nucleotide mutation. Alternatively, themutation is a deletion.

Disease Detection

Disclosed herein are methods of assaying for a target nucleic acid asdescribed herein that can be used for disease detection. For example, amethod of assaying for a target nucleic acid in a sample, comprises:contacting the sample to a complex comprising a guide nucleic acidcomprising a segment that is reverse complementary to a segment of thetarget nucleic acid and a programmable nuclease that exhibits sequenceindependent cleavage upon forming a complex comprising the segment ofthe guide nucleic acid binding to the segment of the target nucleicacid, wherein the sample comprises at least one nucleic acid comprisingat least 50% sequence identity to the segment of the target nucleicacid; and assaying for cleavage of at least one detector nucleic acidsof a population of detector nucleic acids, wherein the cleavageindicates a presence of the target nucleic acid in the sample andwherein absence of the cleavage indicates an absence of the targetnucleic acid in the sample. The detection of the signal can indicate thepresence of the target nucleic acid. Sometimes, the target nucleic acidcomprises a mutation. Often, the mutation is a single nucleotidemutation. Alternatively, the mutation is a deletion. In someembodiments, a method may further comprise administering a treatment forthe disease being detected. Any of the methods described herein may beused in diagnosis, wherein a Cas12 nuclease detects a segment of atarget nucleic acid. Any of the compositions described herein may beused in diagnosis. Any of the programmable nucleases described hereinmay be used in diagnosis, wherein the programmable nuclease detects thetarget nucleic acid.

Also disclosed herein are methods of assaying for a target nucleic acidas described herein that can be used for disease detection. For example,a method of assaying for a target nucleic acid segment in a sample,wherein the target nucleic acid segment lacks a PAM sequence, comprisesamplifying the target nucleic acid segment using a primer having aregion that is reverse complementary to the target nucleic acid segmentand a region that has a PAM sequence reverse complement, therebygenerating a PAM target nucleic acid having a PAM sequence adjacent totarget sequence of an amplification product; contacting the PAM targetnucleic acid to PAM-dependent sequence specific nuclease complexcomprising a guide nucleic acid and a programmable nuclease thatexhibits sequence independent cleavage upon forming a complex comprisingthe segment of the guide nucleic acid binding to the segment of the PAMtarget nucleic acid; and assaying for cleavage of at least one detectornucleic acid of a population of detector nucleic acids, wherein thecleavage indicates a presence of the target nucleic acid in the sampleand wherein the absence of the cleavage indicates an absence of thetarget nucleic acid in the sample. Sometimes, the target nucleic acidcomprises a mutation. Often, the mutation is a single nucleotidemutation.

The methods as described herein can be used to identify or diagnose acancer or genetic disorder associated with a mutation in a targetnucleic acid. The methods can be used to identify a mutation of a targetnucleic acid that affects the expression of a cancer gene. A cancer genecan be any gene whose aberrant expression is associated with cancer,such as overexpression of an oncogene, suppression of tumor suppressorgene, or dysregulation of a checkpoint inhibitor gene or gene associatedwith cellular growth, cellular metabolism, or the cell cycle. A mutationthat affects the expression of cancer gene can be a mutation of a targetnucleic acid within the cancer gene, a mutation of a target nucleic acidcomprising RNA associated with the expression of a cancer gene, or atarget nucleic acid comprising a mutation of a nucleic acid associatedwith regulation of expression of a cancer gene, such as an RNA or apromoter, enhancer, or repressor of the cancer gene. For example, atarget nucleic acid comprising a mutation that affects a cancer gene cancontribute to or lead to colon cancer, bladder cancer, stomach cancer,breast cancer, non-small-cell lung cancer, pancreatic cancer, esophagealcancer, cervical cancer, ovarian cancer, hepatocellular cancer, andacute myeloid leukemia. The target nucleic acid comprise a mutation of acancer gene or RNA expressed from a cancer gene. Often, the mutation isa single nucleotide mutation. Alternatively, the mutation is a deletion.

The methods can be used to identify a mutation that affects theexpression of a gene associated with a genetic disorder. A geneassociated with a genetic disorder can be a gene whose overexpression isassociated with a genetic disorder, from a gene associated with abnormalcellular growth resulting in a genetic disorder, or from a geneassociated with abnormal cellular metabolism resulting in a geneticdisorder. A mutation that affects the expression of a gene associatedwith a genetic disorder can be mutation within the gene associated witha genetic disorder, a mutation of RNA associated with a gene of thegenetic disorder, or a mutation of a nucleic acid associated withregulation of expression of a gene associated with a genetic disorder,such as an RNA or a promoter, enhancer, or repressor of the geneassociated with the genetic disorder. Often, the mutation is a singlenucleotide mutation. Alternatively, the mutation is a deletion.

Methods described herein can be used to identify a mutation in a targetnucleic acid from a bacteria, virus, or microbe. The methods can be usedto identify a mutation of a target nucleic acid that affects theexpression of a gene. A mutation that affects the expression of gene canbe a mutation of a target nucleic acid within the gene, a mutation of atarget nucleic acid comprising RNA associated with the expression of agene, or a target nucleic acid comprising a mutation of a nucleic acidassociated with regulation of expression of a gene, such as an RNA or apromoter, enhancer, or repressor of the gene. Sometimes, a status of atarget nucleic acid mutation is used to determine a pathogenicity of abacteria, virus, or microbe or treatment resistance, such as resistanceto antibiotic treatment. Often, a status of a mutation is used todiagnose or identify diseases associated with the mutation of targetnucleic acid sequences in the bacteria, virus, or microbe. Often, themutation is a single nucleotide mutation. Alternatively, the mutation isa deletion.

Detection as a Research Tool

Disclosed herein are methods of assaying for a target nucleic acid asdescribed herein that can be used as a research tool. For example, amethod of assaying for a target nucleic acid in a sample, comprises:contacting the sample to a complex comprising a guide nucleic acidcomprising a segment that is reverse complementary to a segment of thetarget nucleic acid and a programmable nuclease that exhibits sequenceindependent cleavage upon forming a complex comprising the segment ofthe guide nucleic acid binding to the segment of the target nucleicacid, wherein the sample comprises at least one nucleic acid comprisingat least 50% sequence identity to the segment of the target nucleicacid; and assaying for cleavage of at least one detector nucleic acidsof a population of detector nucleic acids, wherein the cleavageindicates a presence of the target nucleic acid in the sample andwherein absence of the cleavage indicates an absence of the targetnucleic acid in the sample. The detection of the signal can indicate thepresence of the target nucleic acid. Sometimes, the target nucleic acidcomprises a mutation. Often, the mutation is a single nucleotidemutation. Alternatively, the mutation is a deletion.

Also disclosed herein are methods of assaying for a target nucleic acidas described herein that can be used as a research tool. For example, amethod of assaying for a target nucleic acid segment in a sample,wherein the target nucleic acid segment lacks a PAM sequence, comprisesamplifying the target nucleic acid segment using a primer having aregion that is reverse complementary to the target nucleic acid segmentand a region that has a PAM sequence reverse complement, therebygenerating a PAM target nucleic acid having a PAM sequence adjacent totarget sequence of an amplification product; contacting the PAM targetnucleic acid to PAM-dependent sequence specific nuclease complexcomprising a guide nucleic acid and a programmable nuclease thatexhibits sequence independent cleavage upon forming a complex comprisingthe segment of the guide nucleic acid binding to the segment of the PAMtarget nucleic acid; and assaying for cleavage of at least one detectornucleic acid of a population of detector nucleic acids, wherein thecleavage indicates a presence of the target nucleic acid in the sampleand wherein the absence of the cleavage indicates an absence of thetarget nucleic acid in the sample. Sometimes, the target nucleic acidcomprises a mutation. Often, the mutation is a single nucleotidemutation.

The methods as described herein can be used to identify a singlenucleotide mutation in a target nucleic acid. The methods describedherein can be used to identify a deletion in a target nucleic acid. Themethods can be used to identify mutation of a target nucleic acid thataffects the expression of a gene. A mutation that affects the expressionof gene can be a single nucleotide mutation of a target nucleic acidwithin the gene, a mutation of a target nucleic acid comprising RNAassociated with the expression of a gene, or a target nucleic acidcomprising a mutation of a nucleic acid associated with regulation ofexpression of a gene, such as an RNA or a promoter, enhancer, orrepressor of the gene. A mutation that affects the expression of genecan be a deletion of one or more nucleic acids within the gene, adeletion of one or more target nucleic acids comprising RNA associatedwith the expression of a gene, or a target nucleic acid comprising adeletion of one or more nucleic acids associated with regulation ofexpression of a gene, such as an RNA or a promoter, enhancer, orrepressor of the gene. Often, the mutation is a single nucleotidemutation. Alternatively, the mutation is a deletion.

Detection for Agricultural Applications

Disclosed herein are methods of assaying for a target nucleic acid asdescribed herein that can be used for agricultural applications. Forexample, a method of assaying for a target nucleic acid in a sample,comprises: contacting the sample to a complex comprising a guide nucleicacid comprising a segment that is reverse complementary to a segment ofthe target nucleic acid and a programmable nuclease that exhibitssequence independent cleavage upon forming a complex comprising thesegment of the guide nucleic acid binding to the segment of the targetnucleic acid, wherein the sample comprises at least one nucleic acidcomprising at least 50% sequence identity to the segment of the targetnucleic acid; and assaying for cleavage of at least one detector nucleicacids of a population of detector nucleic acids, wherein the cleavageindicates a presence of the target nucleic acid in the sample andwherein absence of the cleavage indicates an absence of the targetnucleic acid in the sample. The detection of the signal can indicate thepresence of the target nucleic acid. Sometimes, the target nucleic acidcomprises a mutation. Often, the mutation is a single nucleotidemutation. Alternatively, the mutation is a deletion.

Also disclosed herein are methods of assaying for a target nucleic acidas described herein that can be used for agricultural applications. Forexample, a method of assaying for a target nucleic acid segment in asample, wherein the target nucleic acid segment lacks a PAM sequence,comprises amplifying the target nucleic acid segment using a primerhaving a region that is reverse complementary to the target nucleic acidsegment and a region that has a PAM sequence reverse complement, therebygenerating a PAM target nucleic acid having a PAM sequence adjacent totarget sequence of an amplification product; contacting the PAM targetnucleic acid to PAM-dependent sequence specific nuclease complexcomprising a guide nucleic acid and a programmable nuclease thatexhibits sequence independent cleavage upon forming a complex comprisingthe segment of the guide nucleic acid binding to the segment of the PAMtarget nucleic acid; and assaying for cleavage of at least one detectornucleic acid of a population of detector nucleic acids, wherein thecleavage indicates a presence of the target nucleic acid in the sampleand wherein the absence of the cleavage indicates an absence of thetarget nucleic acid in the sample. The detection of the signal canindicate the presence of the target nucleic acid. Sometimes, the targetnucleic acid comprises a mutation. Often, the mutation is a singlenucleotide mutation.

The methods as described herein can be used to identify a mutation in atarget nucleic acid of a plant or of a bacteria, virus, or microbeassociated with a plant or soil. The methods can be used to identify amutation of a target nucleic acid that affects the expression of a gene.A mutation that affects the expression of gene can be a mutation of atarget nucleic acid within the gene, a mutation of a target nucleic acidcomprising RNA associated with the expression of a gene, or a targetnucleic acid comprising a mutation of a nucleic acid associated withregulation of expression of a gene, such as an RNA or a promoter,enhancer, or repressor of the gene. Often, the mutation is a singlenucleotide mutation. Alternatively, the mutation is a deletion.

Amplification of Target Nucleic Acids

Disclosed herein are methods of amplifying a target nucleic acid fordetection using any of the methods, reagents, kits or devices describedherein. The compositions for amplification of target nucleic acids andmethods of use thereof, as described herein, are compatible with theDETECTR assay methods disclosed herein. The compositions foramplification of target nucleic acids and methods of use thereof, asdescribed herein, are compatible with any of the programmable nucleasesdisclosed herein (e.g., a programmable nuclease with at least 60%sequence identity to SEQ ID NO: 11) and use of said programmablenuclease in a method of detecting a target nucleic acid. Thecompositions for amplification of target nucleic acids and methods ofuse thereof, as described herein, are compatible with any of thecompositions comprising a programmable nuclease and a buffer, which hasbeen developed to improve the function of the programmable nuclease(e.g., a programmable nuclease and a buffer with low salt (about 110 mMor less) and a pH of 7 to 8) and use of said compositions in a method ofdetecting a target nucleic acid. The compositions for amplification oftarget nucleic acids and methods of use thereof, as described herein,are compatible with any of the methods disclosed herein includingmethods of assaying for at least one base difference (e.g., assaying fora SNP or a base mutation) in a target nucleic acid sequence, methods ofassaying for a target nucleic acid that lacks a PAM by amplifying thetarget nucleic acid sequence to introduce a PAM, and compositions usedin introducing a PAM via amplification into the target nucleic acidsequence. In some cases, amplification of the target nucleic acid mayincrease the sensitivity of a detection reaction. In some cases,amplification of the target nucleic acid may increase the specificity ofa detection reaction. Amplification of the target nucleic acid mayincrease the concentration of the target nucleic acid in the samplerelative to the concentration of nucleic acids that do not correspond tothe target nucleic acid. In some embodiments, amplification of thetarget nucleic acid may be used to modify the sequence of the targetnucleic acid. For example, amplification may be used to insert a PAMsequence into a target nucleic acid that lacks a PAM sequence. In somecases, amplification may be used to increase the homogeneity of a targetnucleic acid sequence. For example, amplification may be used to removea nucleic acid variation that is not of interest in the target nucleicacid sequence.

An amplified target nucleic acid may be present in a DETECTR reaction inan amount relative to an amount of a programmable nuclease. In someembodiments, the amplified target nucleic acid is present in at least1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold,100-fold, 500-fold, 1000-fold, 10,000-fold, or 100,000-fold molar excessrelative to the amount of the programmable nuclease. In someembodiments, the amplified target nucleic acid is present in no morethan 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold,100-fold, 500-fold, 1000-fold, 10,000-fold, or 100,000-fold molar excessrelative to the amount of the programmable nuclease. In someembodiments, the amplified target nucleic acid is present in from 1-foldto 2-fold, from 1-fold to 3-fold, from 1-fold to 4-fold, from 1-fold to5-fold, from 1-fold to 10-fold, from 1-fold to 25-fold, from 1-fold to50-fold, from 1-fold to 100-fold, from 1-fold to 500-fold, from 1-foldto 1000-fold, from 1-fold to 10,000-fold, from 1-fold to 100,000-fold,from 5-fold to 10-fold, from 5-fold to 25-fold, from 5-fold to 50-fold,from 5-fold to 100-fold, from 5-fold to 500-fold, from 5-fold to1000-fold, from 5-fold to 10,000-fold, from 5-fold to 100,000-fold, from10-fold to 25-fold, from 10-fold to 50-fold, from 10-fold to 100-fold,from 10-fold to 500-fold, from 10-fold to 1000-fold, from 10-fold to10,000-fold, from 10-fold to 100,000-fold, from 100-fold to 500-fold,from 100-fold to 1000-fold, from 100-fold to 10,000-fold, from 100-foldto 100,000-fold, from 1000-fold to 10,000-fold, from 1000-fold to100,000-fold, or from 10,000-fold to 100,000-fold molar excess relativeto the amount of the programmable nuclease. In some embodiments, theprogrammable nuclease is present in at least 1-fold, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 500-fold,1000-fold, 10,000-fold, or 100,000-fold molar excess relative to theamount of the target nucleic acid. In some embodiments, the programmablenuclease is present in no more than 1-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 500-fold, 1000-fold,10,000-fold, or 100,000-fold molar excess relative to the amount of thetarget nucleic acid. In some embodiments, the programmable nuclease ispresent in from 1-fold to 2-fold, from 1-fold to 3-fold, from 1-fold to4-fold, from 1-fold to 5-fold, from 1-fold to 10-fold, from 1-fold to25-fold, from 1-fold to 50-fold, from 1-fold to 100-fold, from 1-fold to500-fold, from 1-fold to 1000-fold, from 1-fold to 10,000-fold, from1-fold to 100,000-fold, from 5-fold to 10-fold, from 5-fold to 25-fold,from 5-fold to 50-fold, from 5-fold to 100-fold, from 5-fold to500-fold, from 5-fold to 1000-fold, from 5-fold to 10,000-fold, from5-fold to 100,000-fold, from 10-fold to 25-fold, from 10-fold to50-fold, from 10-fold to 100-fold, from 10-fold to 500-fold, from10-fold to 1000-fold, from 10-fold to 10,000-fold, from 10-fold to100,000-fold, from 100-fold to 500-fold, from 100-fold to 1000-fold,from 100-fold to 10,000-fold, from 100-fold to 100,000-fold, from1000-fold to 10,000-fold, from 1000-fold to 100,000-fold, or from10,000-fold to 100,000-fold molar excess relative to the amount of thetarget nucleic acid. In some embodiments, the target nucleic acid is notpresent in the sample.

An amplified target nucleic acid may be present in a DETECTR reaction inan amount relative to an amount of a guide nucleic acid. In someembodiments, the amplified target nucleic acid is present in at least1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold,100-fold, 500-fold, 1000-fold, 10,000-fold, or 100,000-fold molar excessrelative to the amount of the guide nucleic acid. In some embodiments,the amplified target nucleic acid is present in no more than 1-fold,2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold,500-fold, 1000-fold, 10,000-fold, or 100,000-fold molar excess relativeto the amount of the guide nucleic acid. In some embodiments, theamplified target nucleic acid is present in from 1-fold to 2-fold, from1-fold to 3-fold, from 1-fold to 4-fold, from 1-fold to 5-fold, from1-fold to 10-fold, from 1-fold to 25-fold, from 1-fold to 50-fold, from1-fold to 100-fold, from 1-fold to 500-fold, from 1-fold to 1000-fold,from 1-fold to 10,000-fold, from 1-fold to 100,000-fold, from 5-fold to10-fold, from 5-fold to 25-fold, from 5-fold to 50-fold, from 5-fold to100-fold, from 5-fold to 500-fold, from 5-fold to 1000-fold, from 5-foldto 10,000-fold, from 5-fold to 100,000-fold, from 10-fold to 25-fold,from 10-fold to 50-fold, from 10-fold to 100-fold, from 10-fold to500-fold, from 10-fold to 1000-fold, from 10-fold to 10,000-fold, from10-fold to 100,000-fold, from 100-fold to 500-fold, from 100-fold to1000-fold, from 100-fold to 10,000-fold, from 100-fold to 100,000-fold,from 1000-fold to 10,000-fold, from 1000-fold to 100,000-fold, or from10,000-fold to 100,000-fold molar excess relative to the amount of theguide nucleic acid. In some embodiments, the guide nucleic acid ispresent in at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold,25-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 10,000-fold, or100,000-fold molar excess relative to the amount of the target nucleicacid. In some embodiments, the guide nucleic acid is present in no morethan 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold,100-fold, 500-fold, 1000-fold, 10,000-fold, or 100,000-fold molar excessrelative to the amount of the target nucleic acid. In some embodiments,the guide nucleic acid is present in from 1-fold to 2-fold, from 1-foldto 3-fold, from 1-fold to 4-fold, from 1-fold to 5-fold, from 1-fold to10-fold, from 1-fold to 25-fold, from 1-fold to 50-fold, from 1-fold to100-fold, from 1-fold to 500-fold, from 1-fold to 1000-fold, from 1-foldto 10,000-fold, from 1-fold to 100,000-fold, from 5-fold to 10-fold,from 5-fold to 25-fold, from 5-fold to 50-fold, from 5-fold to 100-fold,from 5-fold to 500-fold, from 5-fold to 1000-fold, from 5-fold to10,000-fold, from 5-fold to 100,000-fold, from 10-fold to 25-fold, from10-fold to 50-fold, from 10-fold to 100-fold, from 10-fold to 500-fold,from 10-fold to 1000-fold, from 10-fold to 10,000-fold, from 10-fold to100,000-fold, from 100-fold to 500-fold, from 100-fold to 1000-fold,from 100-fold to 10,000-fold, from 100-fold to 100,000-fold, from1000-fold to 10,000-fold, from 1000-fold to 100,000-fold, or from10,000-fold to 100,000-fold molar excess relative to the amount of thetarget nucleic acid. In some embodiments, the target nucleic acid is notpresent in the sample.

Amplification for Insertion of a PAM Sequence

Amplification methods can also enhance the assay detection of the targetnucleic acid, such as enhancing a method of assaying for a targetnucleic acid in a sample, comprises: contacting the sample to a complexcomprising a guide nucleic acid comprising a segment that is reversecomplementary to a segment of the target nucleic acid and a programmablenuclease that exhibits sequence independent cleavage upon forming acomplex comprising the segment of the guide nucleic acid binding to thesegment of the target nucleic acid, wherein the sample comprises atleast one nucleic acid comprising at least 50% sequence identity to thesegment of the target nucleic acid; and assaying for cleavage of atleast one detector nucleic acids of a population of detector nucleicacids, wherein the cleavage indicates a presence of the target nucleicacid in the sample and wherein absence of the cleavage indicates anabsence of the target nucleic acid in the sample. For example,amplification of a target nucleic acid with primers encoding a PAMsequence to insert the PAM sequence into the sequence of the targetnucleic acid before the contacting. More specifically, a PAM targetnucleic acid comprising a sequence encoding a PAM sequence (e.g., TTTNor dUdUdUN) is produced by amplifying the target nucleic acid segmentusing a primer having a region that is reverse complementary to thetarget nucleic acid segment and a region that has a PAM sequence reversecomplement, thereby generating a PAM target nucleic acid having a PAMsequence adjacent to target sequence of an amplification product. Often,a sequence encoding a PAM sequence is TTTN. Sometimes, a sequenceencoding a PAM is dUdUdUN. This allows for any target nucleic acid to beused with a programmable nuclease (e.g., Cas12) that requires the targetnucleic acid to comprise a sequence encoding a PAM for activation of theprogrammable nuclease complexed with the guide nucleic acid. This allowsfor any target nucleic acid to be used with a programmable nuclease(e.g., Cas12) that requires the target nucleic acid to comprise asequence encoding a PAM for binding to the guide nucleic acid. One ormore steps of the method as disclosed herein may be performed in acommon reaction volume (e.g., a single reaction mixture). Often, themethod as disclosed herein is performed in a common reaction volume.

Often, the primer is a forward primer. For example, the forward primercomprises the sequence encoding the PAM. Sometimes, the forward primercomprises from 1 to 20 nucleotides from the 3′ end of the sequenceencoding the PAM. Often, the forward primer comprises from 1 to 8nucleotides from the 3′ end of the sequence encoding the PAM. Theforward primer can comprise 6 nucleotides from the 3′ end of thesequence encoding the PAM. The forward primer can comprise 7 nucleotidesfrom the 3′ end of the sequence encoding the PAM. The forward primer cancomprise 8 nucleotides from the 3′ end of the sequence encoding the PAM.Sometimes, these nucleotides from the 3′ end of the sequence encodingthe PAM is referred are referred to extension nucleotides (e.g., 6nucleotide extension).

Often, a mutation in the target nucleic acid amplified using the primeris located a certain number of nucleotides downstream of the 5′ end ofthe target nucleic acid segment wherein the target nucleic acid segmentis a segment that binds to a segment of the guide nucleic acid that isreverse complementary to it and comprises the sequence encoding the PAM.Sometimes, the mutation is a single nucleotide mutation or a SNP (e.g.,a synonymous mutation or a non-synonymous mutation such as a missensesubstitution or a nonsense point mutation). Sometimes, the mutation is adeletion. Often, the mutation is from 3 to 20 nucleotides downstream ofthe target nucleic acid segment. Sometimes, the mutation is from 5 to 9nucleotides downstream of the target nucleic acid segment. The mutationcan be 6 nucleotides downstream of the target nucleic acid segment. Themutation can be 7 nucleotides downstream of the target nucleic acidsegment. The mutation can be 8 nucleotides downstream of the targetnucleic acid segment.

A method of assaying for a target nucleic acid segment in a sample,wherein the target nucleic acid segment lacks a PAM sequence, comprisesamplifying the target nucleic acid segment using a primer having aregion that is reverse complementary to the target nucleic acid segmentand a region that has a PAM sequence reverse complement, therebygenerating a PAM target nucleic acid having a PAM sequence adjacent totarget sequence of an amplification product; contacting the PAM targetnucleic acid to PAM-dependent sequence specific nuclease complexcomprising a guide nucleic acid and a programmable nuclease thatexhibits sequence independent cleavage upon forming a complex comprisingthe segment of the guide nucleic acid binding to the segment of the PAMtarget nucleic acid; and assaying for cleavage of at least one detectornucleic acid of a population of detector nucleic acids, wherein thecleavage indicates a presence of the target nucleic acid in the sampleand wherein the absence of the cleavage indicates an absence of thetarget nucleic acid in the sample. Often, a sequence encoding a PAMsequence is TTTN. Sometimes, a sequence encoding a PAM is dUdUdUN. Thisallows for any target nucleic acid to be used with a programmablenuclease (e.g., Cas12) that requires the target nucleic acid to comprisea sequence encoding a PAM for activation of the programmable nucleasecomplexed with the guide nucleic acid. One or more steps of the methodas disclosed herein may be performed in a common reaction volume (e.g.,a single reaction mixture). Often, the method as disclosed herein isperformed in a common reaction volume.

Often, the forward primer comprises the sequence encoding the PAM.Sometimes, the PAM forward primer comprises from 1 to 20 nucleotidesfrom the 3′ end of the sequence encoding the PAM. Often, the PAM forwardprimer comprises from 1 to 8 nucleotides from the 3′ end of the sequenceencoding the PAM. Sometimes, the PAM forward primer comprises from 1 to2 or 4 to 8 nucleotides from the 3′ end of the sequence encoding thePAM. Often a PAM forward primer comprising from 1 to 2 or 4 to 8nucleotides from the 3′ end of the sequence encoding the PAM is a PAMsequence comprising dUdUdUN. The PAM forward primer can comprise 1nucleotides from the 3′ end of the sequence encoding the PAM. The PAMforward primer can comprise 2 nucleotides from the 3′ end of thesequence encoding the PAM. The PAM forward primer can comprise 3nucleotides from the 3′ end of the sequence encoding the PAM. The PAMforward primer can comprise 4 nucleotides from the 3′ end of thesequence encoding the PAM. The PAM forward primer can comprise 5nucleotides from the 3′ end of the sequence encoding the PAM. The PAMforward primer can comprise 6 nucleotides from the 3′ end of thesequence encoding the PAM. The PAM forward primer can comprise 7nucleotides from the 3′ end of the sequence encoding the PAM. The PAMforward primer can comprise 8 nucleotides from the 3′ end of thesequence encoding the PAM. Sometimes, these nucleotides from the 3′ endof the sequence encoding the PAM is referred are referred to extensionnucleotides (e.g., 6 nucleotide extension).

Often, a mutation in the target nucleic acid (also referred to as themismatch) amplified using PAM primers is located a certain number ofnucleotides downstream of the 5′ end of the PAM in PAM target nucleicacid. Sometimes, the mutation or mismatch is a single nucleotidemutation or a SNP. Often, the mismatch is from 3 to 20 nucleotidesdownstream of the PAM in PAM target nucleic acid. The mismatch can befrom 3 to 10 nucleotides downstream of the PAM in PAM target nucleicacid. Sometimes, the mismatch is from 5 to 9 nucleotides downstream ofthe PAM in PAM target nucleic acid. The mutation can be 6 nucleotidesdownstream of the PAM in PAM target nucleic acid. The mutation can be 7nucleotides downstream of the PAM in PAM target nucleic acid. Themutation can be 8 nucleotides downstream of the PAM in PAM targetnucleic acid.

The amplification that produces the PAM target nucleic acid can beperformed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes. Sometimes,the amplification reaction is performed at a temperature of around20-45° C. The amplification reaction can be performed at a temperatureno greater than 20° C., 25° C., 30° C., 35° C., 37° C., 40° C., 45° C.The reaction can be performed at a temperature of at least 20° C., 25°C., 30° C., 35° C., 37° C., 40° C., or 45° C. Sometimes, theamplification is performed with dTTP, dATP, dCTP, and dGTP. Often theamplification is performed with dUTP, dATP, dCTP, and dGTP. In someembodiments, an amplified target nucleic acid comprises dU nucleicacids.

The amplification that produces the PAM target nucleic acid can bethermal cycling amplification or isothermal amplification. The reagentsfor the amplification can comprise a recombinase, a oligonucleotideprimer, a single-stranded DNA binding (SSB) protein, and a polymerase.The isothermal amplification can be transcription mediated amplification(TMA). Isothermal amplification can be helicase dependent amplification(HDA) or circular helicase dependent amplification (cHDA). In additionalcases, isothermal amplification is strand displacement amplification(SDA). The isothermal amplification can be recombinase polymeraseamplification (RPA). The isothermal amplification can be at least one ofloop mediated amplification (LAMP) or the exponential amplificationreaction (EXPAR). Isothermal amplification is, in some cases, by rollingcircle amplification (RCA), ligase chain reaction (LCR), simple methodamplifying RNA targets (SMART), single primer isothermal amplification(SPIA), multiple displacement amplification (MDA), nucleic acid sequencebased amplification (NASBA), hinge-initiated primer-dependentamplification of nucleic acids (HIP), nicking enzyme amplificationreaction (NEAR), or improved multiple displacement amplification (IMDA).In a preferred embodiment, the isothermal amplification is LAMP.

Various compositions are compatible with the amplification methodsdescribed herein. In some embodiments, a composition may comprise anucleic acid from a sample, wherein the nucleic acid comprises a PAM anda segment that hybridizes to a guide nucleic acid, wherein the PAM has asequence of dUdUdUN, a guide nucleic acid that hybridizes to the segmentof the nucleic acid, and a programmable nuclease that exhibits sequenceindependent cleavage of a detector nucleic acid upon hybridization ofthe guide nucleic acid to the segment of the target nucleic acid. Acomposition may further comprise a primer, wherein the primer comprisesa first region that is reverse complementary to the PAM and a secondregion that is reverse complementary to a first segment of the nucleicacid.

Various methods of assaying are compatible with the amplificationmethods described herein. In some embodiments, a method of assaying fora target nucleic acid in a sample, wherein the target nucleic acid lacksa PAM may comprise amplifying the target nucleic acid from a sampleusing a primer comprising a first region that is reverse complementaryto a PAM and a second region that is reverse complementary to a firstsegment of the target nucleic acid, wherein the PAM is dUdUdUN, therebyproducing a PAM target nucleic acid, contacting the PAM target nucleicacid to a guide nucleic acid that hybridizes to a segment of the PAMtarget nucleic acid, a programmable nuclease that exhibits sequenceindependent cleavage of a detector nucleic acid upon hybridization ofthe guide nucleic acid to a segment of the PAM target nucleic acid, anda detector nucleic acid, and assaying for a signal produced by cleavageof the detector nucleic acid. In some embodiments, the second regioncomprises from 4 to 12 bases. In some embodiments, the second regioncomprises from 4 to 10 bases. In some embodiments, the second regioncomprises from 4 to 7 bases.

Amplification Using Blocking Primer

Amplification methods can also enhance the assay detection of the targetnucleic acid, such as enhancing a method of assaying for a targetnucleic acid in a sample, comprises: contacting the sample to a complexcomprising a guide nucleic acid comprising a segment that is reversecomplementary to a segment of the target nucleic acid and a programmablenuclease that exhibits sequence independent cleavage upon forming acomplex comprising the segment of the guide nucleic acid binding to thesegment of the target nucleic acid, wherein the sample comprises atleast one nucleic acid comprising at least 50% sequence identity to thesegment of the target nucleic acid; and assaying for cleavage of atleast one detector nucleic acids of a population of detector nucleicacids, wherein the cleavage indicates a presence of the target nucleicacid in the sample and wherein absence of the cleavage indicates anabsence of the target nucleic acid in the sample.

The methods described herein may comprise amplifying the target prior todetection. In some embodiments, amplifying may comprise using a blockingprimer. In some cases, amplification may be performed in the presence ofa blocking primer to block amplification of a nucleic acid sequencecomprising a mutation or a variation relative to the target nucleicacid. The mutation can be a single nucleotide mutation, a SNP, or adeletion. The variant can be the wild type variant of the mutation(e.g., the wild type variant of the single nucleotide mutation or thewild type variant of the SNP). For example, the blocking primer may bindto a nucleic acid region comprising the mutation relative to the targetnucleic acid but may not bind to the target nucleic acid that does notcomprise the mutation. In some embodiments, the blocking primer binds toa nucleic acid comprising encoding the wild type sequence of the targetnucleic acid segment. Binding of the blocking primer to the nucleic acidregion comprising the mutation may prevent amplification of the nucleicacid sequence comprising the mutation. Often, the blocking primercomprises a 3′ phosphate. The blocking primer may be a primer incapableof initiating nucleic acid extension. The blocking primer may preventbinding of a primer that is capable of initiating nucleic acidextension. In some cases, the blocking primer can bind perfectly to thenucleic acid comprising the variant mutation. Amplification in thepresence of the blocking primer may be performed before the contactingof the methods described herein.

The use of a blocking primer results in selective amplification of thetarget nucleic acid. This occurs using standard PCR conditions when ablocking primer is added with a forward primer and a reverse primer. Theblocking primer and either the forward or the reverse primer encode atleast part of a sequence that overlaps with the sequence of the blockingprimer. In this PCR reaction, the blocking primer binds to a variant ofthe mutation of the target nucleic acid and blocks either the forwardprimer or the reverse primer (depending on which primer comprises theoverlapping sequence with the blocking primer) from priming theextension of the nucleic acid comprising variant of the mutation of thetarget nucleic acid, and thus the nucleic acid comprising the variant ofthe mutation of the target nucleic acid is not amplified. In contrast,the blocking primer does not bind the mutation of the target nucleicacid and does not block either the forward primer or the reverse primer(depending on which primer comprises the overlapping sequence with theblocking primer) from priming the extension of the nucleic acidcomprising variant of the mutation of the target nucleic acid, and thusthe target nucleic acid is selectively amplified. This results in targetnucleic acid enrichment in the before the contacting step of the methodsdescribed herein.

COLD-PCR Amplification

Amplification methods can also enhance the assay detection of the targetnucleic acid, such as enhancing a method of assaying for a targetnucleic acid in a sample, comprises: contacting the sample to a complexcomprising a guide nucleic acid comprising a segment that is reversecomplementary to a segment of the target nucleic acid and a programmablenuclease that exhibits sequence independent cleavage upon forming acomplex comprising the segment of the guide nucleic acid binding to thesegment of the target nucleic acid, wherein the sample comprises atleast one nucleic acid comprising at least 50% sequence identity to thesegment of the target nucleic acid; and assaying for cleavage of atleast one detector nucleic acids of a population of detector nucleicacids, wherein the cleavage indicates a presence of the target nucleicacid in the sample and wherein absence of the cleavage indicates anabsence of the target nucleic acid in the sample. For example,amplification is performed using co-amplification at lower denaturationtemperature PCR (COLD-PCR), such as full COLD-PCR and fast COLD-PCR,before the contacting of the methods described herein. In someembodiments, amplifying comprises fast COLD-PCR. In some embodiments,amplifying comprises allele-specific COLD-PCR. In some embodiments,amplifying comprises COLD-PCR. Often, the target nucleic acid is from0.05% to 20% of total nucleic acids in the sample in these methods.

The mismatches from mutations in the segment of the target nucleic acid,such as a single nucleotide mutation or a deletion, compared to anucleic acid comprising at least 50% sequence identity to the segment ofthe target nucleic acid, result in altering the melting temperature (Tm)of a double stranded DNA comprising the segment of the target nucleicacid. For example, a target nucleic acid comprising the segment of thetarget nucleic acid has a Tm that is from 0.1 to 5 C lower than thenucleic acid comprising at least 50% sequence identity to segment of thetarget nucleic acid. Both full COLD-PCR and fast COLD-PCR are based onthis principle and can be used to selectively amplify the target nucleicacid comprising the mutation.

For performing amplification using full COLD-PCR, the sample comprisingthe segment of the target nucleic acid and nucleic acid comprising atleast 50% sequence identity to the segment of the target nucleic acidcan undergo a denaturation step, such as denaturation at hightemperature (e.g., about 94° C. or higher). Next, the temperature ischanged to an intermediate annealing temperature that allowshybridization of the segment of the target nucleic acid and the nucleicacid comprising at least 50% sequence identity to the segment of thetarget nucleic acid to one another. After hybridization, theheteroduplexes of the segment of the target nucleic acid and the nucleicacid comprising at least 50% sequence identity to the segment of thetarget nucleic acid melt at lower temperatures for denaturation (at a Tctemperature which is a critical temperature of the double stranded DNAthat is lower than its Tm) while the homoduplexes of the segment of thetarget nucleic acid or the homoduplexes of the nucleic acid comprisingat least 50% sequence identity to the segment of the target nucleic acidremain double stranded. Mismatched sequences (e.g., heteroduplexes ofthe segment of the target nucleic acid and the nucleic acid comprisingat least 50% sequence identity to the segment of the target nucleicacid) may be selectively denatured at a critical temperature (“Tc,”e.g., about 86.5° C.). Matched sequences (e.g., homoduplexes of thesegment of the target nucleic acid or the homoduplexes of the nucleicacid comprising at least 50% sequence identity to the segment of thetarget nucleic acid) may remain double stranded during selectivedenaturation of the mismatched sequences. Primers can then anneal to thedenatured strands and a DNA polymerase can extend these strands. Sinceonly heteroduplexes of the segment of the target nucleic acid and thenucleic acid comprising at least 50% sequence identity to the segment ofthe target nucleic acid are available for amplification, a largerportion of the target nucleic acid is amplified and also becomesavailable for amplification is subsequent rounds. FIG. 64A illustratesan exemplary protocol of full COLD-PCR.

For performing amplification using fast COLD-PCR, the Tm of the segmentof the target nucleic acid is lower than the Tm of the nucleic acidcomprising at least 50% sequence identity to the segment of the targetnucleic acid. Thus, fast COLD-PCR can enrich for segment of the targetnucleic acids comprising a mutation that results in a lower Tm than theTm of the nucleic acid comprising at least 50% sequence identity to thesegment of the target nucleic acid. For fast COLD-PCR, the samplecomprising the segment of the target nucleic acid and nucleic acidcomprising at least 50% sequence identity to the segment of the targetnucleic acid can undergo a denaturation step, such as denaturation athigh temperature (e.g., 94° C.). Next, the temperature is reduced sothat primers can then anneal to the denatured strands of the segment ofthe target nucleic acid and a DNA polymerase can extend these strands.Since the segment of the target nucleic acid can be denatured at a lowertemperature than the nucleic acid comprising at least 50% sequenceidentity to the segment of the target nucleic acid, the segment of thetarget nucleic acid is amplified while the nucleic acid comprising atleast 50% sequence identity to segment of the target nucleic acidremains double stranded. Mutant sequences (e.g., the segment of thetarget nucleic acid comprising a mutation) may be selectively denaturedat a critical temperature (“Tc,” e.g., about 86.5° C.). Wild typesequences (e.g., the nucleic acid comprising at least 50% sequenceidentity to the segment of the target nucleic acid) may remain doublestranded during selective denaturation of the mutant sequences. FIG. 64Billustrates an exemplary protocol of fast COLD-PCR.

In some embodiments, a composition comprising a Cas12 programmablenuclease (e.g., SEQ ID NO: 11) is at a temperature of from 25° C. to 45°C. The Cas12 programmable nuclease (e.g., SEQ ID NO: 11) may exhibitcatalytic activity at a temperature of from 25° C. to 45° C. The Cas12programmable nuclease (e.g., SEQ ID NO: 11) may exhibit catalyticactivity after heating the composition to a temperature of greater than45° C. and restoring the temperature to a temperature of from 25° C. to45° C.

Allele Specific PCR Amplification

Amplification methods can also enhance the assay detection of the targetnucleic acid, such as enhancing a method of assaying for a targetnucleic acid in a sample, comprises: contacting the sample to a complexcomprising a guide nucleic acid comprising a segment that is reversecomplementary to a segment of the target nucleic acid and a programmablenuclease that exhibits sequence independent cleavage upon forming acomplex comprising the segment of the guide nucleic acid binding to thesegment of the target nucleic acid, wherein the sample comprises atleast one nucleic acid comprising at least 50% sequence identity to thesegment of the target nucleic acid; and assaying for cleavage of atleast one detector nucleic acids of a population of detector nucleicacids, wherein the cleavage indicates a presence of the target nucleicacid in the sample and wherein absence of the cleavage indicates anabsence of the target nucleic acid in the sample. For example,amplification is performed using allele-specific PCR. Allele-specificPCR comprises using a common reverse primer and two forwardallele-specific primers with different 3′ ends to amplify the twoallele-specific PCR products of different lengths. Often the forwardprimer for the segment of the target nucleic acid comprises the mutationat the 3′ end of the primer and the forward primer for the nucleic acidcomprising at least 50% sequence identity segment of the to the targetnucleic acid comprises a variant of the mutation at the 3′ end of theprimer. The 3′ end can cause a mismatch that will result in the primernot functioning as a primer under appropriate conditions. This allowsfor the choosing of conditions that allow for the amplification of thesegment of the target nucleic acid but not the nucleic acid comprisingat least 50% sequence identity to the segment of the target nucleicacid. Often, the products from the two different forward primers arealso different lengths, so these two products can be separated based ontheir differing lengths using techniques, such as agarose gelelectrophoresis. Therefore, the segment of the target nucleic can beenriched before the contacting in the method described herein.

Often, allele-specific PCR is combined with COLD-PCR. Sometimes,allele-specific PCR is combined with full COLD-PCR as described above.Sometimes, allele-specific PCR is combined with fast COLD-PCR asdescribed above.

Primer and Guide Nucleic Acid Design for Amplification and Detection

A number of target amplification and detection methods are consistentwith the methods, compositions, reagents, enzymes, and kits disclosedherein. The target amplification and detection methods, as describedherein, are compatible with the DETECTR assay methods disclosed herein.The target amplification and detection methods, as described herein, arecompatible with any of the programmable nucleases disclosed herein(e.g., a programmable nuclease with at least 60% sequence identity toSEQ ID NO: 11) and use of said programmable nuclease in a method ofdetecting a target nucleic acid. The target amplification and detectionmethods, as described herein, are compatible with any of thecompositions comprising a programmable nuclease and a buffer, which hasbeen developed to improve the function of the programmable nuclease(e.g., a programmable nuclease and a buffer with low salt (about 110 mMor less) and a pH of 7 to 8) and use of said compositions in a method ofdetecting a target nucleic acid. The target amplification and detectionmethods, as described herein, are compatible with any of the methodsdisclosed herein including methods of assaying for at least one basedifference (e.g., assaying for a SNP or a base mutation) in a targetnucleic acid sequence, methods of assaying for a target nucleic acidthat lacks a PAM by amplifying the target nucleic acid sequence tointroduce a PAM, and compositions used in introducing a PAM viaamplification into the target nucleic acid sequence. As describedherein, a target nucleic acid may be detected using a DNA-activatedprogrammable RNA nuclease (e.g., a Cas13), a DNA-activated programmableDNA nuclease (e.g., a Cas12), or an RNA-activated programmable RNAnuclease (e.g., a Cas13) and other reagents disclosed herein (e.g., RNAcomponents). The target nucleic acid may be detected using DETECTR, asdescribed herein. The target nucleic acid may be an RNA, reversetranscribed RNA, DNA, DNA amplicon, amplified DNA, synthetic nucleicacids, or nucleic acids found in biological or environmental samples.Amplification methods can also enhance the assay detection of the targetnucleic acid sequence, such as enhancing a method of assaying for atarget nucleic acid in a sample. In some cases, the target nucleic acidis amplified prior to or concurrent with detection. In some cases, thetarget nucleic acid is reverse transcribed prior to amplification. Thetarget nucleic acid may be amplified via loop mediated isothermalamplification (LAMP) of a target nucleic acid sequence. In some cases,the nucleic acid is amplified using LAMP coupled with reversetranscription (RT-LAMP). The LAMP amplification may be performedindependently, or the LAMP amplification may be coupled to DETECTR fordetection of the target nucleic acid. The RT-LAMP amplification may beperformed independently, or the RT-LAMP amplification may be coupled toDETECTR for detection of the target nucleic acid. The DETECTR reactionmay be performed using any method consistent with the methods disclosedherein.

Amplification and Detection Reaction Mixtures

In some embodiments, a LAMP amplification reaction comprises a pluralityof primers, dNTPs, and a DNA polymerase. LAMP may be used to amplify DNAwith high specificity under isothermal conditions. The DNA may be singlestranded DNA or double stranded DNA. In some cases, a target nucleicacid comprising RNA may be reverse transcribed into DNA using a reversetranscriptase prior to LAMP amplification. A reverse transcriptionreaction may comprise primers, dNTPs, and a reverse transcriptase. Insome cases, the reverse transcription reaction and the LAMPamplification reaction may be performed in the same reaction. A combinedRT-LAMP reaction may comprise LAMP primers, reverse transcriptionprimers, dNTPs, a reverse transcriptase, and a DNA polymerase. In somecase, the LAMP primers may comprise the reverse transcription primers.In some embodiments, the dNTPs may comprise dTTP, dATP, dGTP, and dCTP.In some embodiments, the dNTPs may comprise dUTP, dATP, dGTP, and dCTP.

A target nucleic acid may be reverse transcribed prior to or concurrentwith amplification. For example, an RNA target nucleic acid may bereverse transcribed into DNA. A reverse transcription reaction maycomprise an RNA target nucleic acid, dNTPs, and a reverse transcriptase.In some embodiments, the dNTPs may comprise dTTP, dATP, dGTP, and dCTP.In some embodiments, the dNTPs may comprise dUTP, dATP, dGTP, and dCTP.Reverse transcription may be performed in the same reaction as LAMPamplification as a reverse transcription LAMP (RT-LAMP reaction). Anamplified target nucleic acid may be transcribed using in vitrotranscription (IVT) concurrent with or subsequent to amplification. Theamplification may be LAMP, or the amplification may be RT-LAMP. An IVTreaction may comprise an amplified target nucleic acid, NTPs, and an RNApolymerase. In some embodiments, the amplified target nucleic acidcomprises dU nucleic acids.

In some embodiments, an amplification reaction comprises an uracil-DNAglycosylase (UDG) enzyme. The UDG enzyme may be heat-activated (e.g., atabout 50° C.) to degrade any nucleic acid containing dU in the sample.For example, the heat-activated UDG enzyme may degrade contaminating DNAcontaining dU. The UDG enzyme may be heat-inactivated (e.g., at 95° C.)after degradation of the nucleic acid containing dU and prior toamplification of the target nucleic acid. For example, theheat-inactivated UDG enzyme may be inactivated prior to amplifying atarget nucleic acid sequence using dNTPs comprising dUTP. An active UDGenzyme may be added to an amplification reaction prior to amplificationto degrade contaminating nucleic acids containing dU. In someembodiments, the UDG enzyme is removed prior to amplification of thetarget nucleic acid. The UDG enzyme may also be present in an inactivestate during amplification of the target nucleic acid using dUTPs. Insome embodiments, active UDG enzyme is present in an amplificationreaction using dNTPs that do not comprise dUTP.

A DETECTR reaction to detect the target nucleic acid sequence maycomprise a guide nucleic acid comprising a segment that is reversecomplementary to a segment of the target nucleic acid and a programmablenuclease. The programmable nuclease when activated, as describedelsewhere herein, exhibits sequence-independent cleavage of a reporter(e.g., a nucleic acid comprising a moiety that becomes detectable uponcleavage of the nucleic acid by the programmable nuclease). Theprogrammable nuclease is activated upon the guide nucleic acidhybridizing to the target nucleic acid. In some embodiments, the targetnucleic acid comprises dU nucleic acids. A combined LAMP DETECTRreaction may comprise a plurality of primers, dNTPs, a DNA polymerase, aguide nucleic acid, a programmable nuclease, and a substrate nucleicacid. A combined RT-LAMP DETECTR reaction may comprise LAMP primers,reverse transcription primers, dNTPs, a reverse transcriptase, a DNApolymerase, a guide nucleic acid, a programmable nuclease, and asubstrate nucleic acid. In some case, the LAMP primers may comprise thereverse transcription primers. LAMP and DETECTR can be carried out inthe same sample volume. LAMP and DETECTR can be carried out concurrentlyin separate sample volumes or in the same sample volume. RT-LAMP andDETECTR can be carried out in the same sample volume. RT-LAMP andDETECTR can be carried out concurrently in separate sample volumes or inthe same sample volume.

Primer Design for LAMP Amplification

A LAMP reaction may comprise a plurality of primers. A plurality ofprimers are designed to amplify a target nucleic acid sequence, which isshown in FIG. 40 relative to various regions of a double strandednucleic acid. The primers can anneal to or have sequences correspondingto these various regions. As shown in FIG. 40, the F1c region is 5′ ofthe F2c region, and the F2c region is 5′ of the F3c region.Additionally, the B1 region is 3′ of the B2 region, and the B2 region is3′ of the B3 region. The F3c, F2c, F1c, B1, B2, and B3 regions are shownon the lower strand in FIG. 40. An F3 region is a sequence reversecomplementary to the F3c region. An F2 region is a sequence reversecomplementary to the F2c region. An F1 region is a sequence reversecomplementary to the F1c region. The B1c region is a sequence reversecomplementary to a B1 region. The B2c region is a sequence reversecomplementary to a B2 region. The B3c region is a sequence reversecomplementary to a B3 region. The target nucleic acid may be 5′ of theF1c region and 3′ of the B1 region, as shown in the top configuration ofFIG. 40. The target nucleic acid may be 5′ of the B1c region and 3′ ofthe F1 region, as shown in the bottom configuration of FIG. 40. In someembodiments, the target nucleic acid may be 5′ of the F2c region and 3′of the F1c region. In some embodiments, the target nucleic acid may be5′ of the B2c region and 3′ of the B1c region. In some embodiments, thetarget nucleic acid sequence may be 5′ of the B1 region and 3′ of the B2region. In some embodiments, the target nucleic acid sequence may be 5′of the F1 region and 3′ of the F2 region.

FIG. 40 also shows the structure and directionality of the variousprimers. The forward outer primer has a sequence of the F3 region. Thus,the forward outer primer anneals to the F3c region. The backward outerprimer has a sequence of the B3 region. Thus, the backward outer primeranneals to the B3c region. The forward inner primer has a sequence ofthe F1c region 5′ of a sequence of the F2 region. Thus, the F2 region ofthe forward inner primer anneals to the F2c region and the amplifiedsequence forms a loop held together via hybridization of the sequence ofthe F1c region in the forward inner primer and the F1. The backwardinner primer has a sequence of a B1c region 5′ of a sequence of the B2region. Thus, the B2 region of the backward inner primer anneals to theB2c region and the amplified sequence forms a loop held together viahybridization of the sequence of the B1c region of the backward innerprimer and the B1 region.

Further, as shown in FIG. 40, the plurality of primers may additionallyinclude a loop forward primer (LF) and/or a loop backward primer (LB).LF is positioned 3′ of the F1c region and 5′ of the F2c region. LB ispositioned 5′ of the B2c region and 3′ of the B1c region. The F1, F1c,F2, F2c, F3, F3c, B1, B1c, B2, B2c, B3, and/or B3c regions areillustrated in various arrangements relative to the target nucleic acid,the PAM, and the guide RNA (gRNA), as shown in any one of FIG. 40-FIG.42 or FIG. 50-FIG. 51. The target nucleic acid may be within the nucleicacid strand comprising the B1, B2, B3, LF, F1c, F2c, F3c, and LBcregions. The target nucleic acid may be within the nucleic acid strandcomprising the F1, F2, F3, LB, B1c, B2c, B3c, and LFc regions.

A set of LAMP primers may be designed for use in combination with aDETECTR reaction. The nucleic acid may comprise a region (e.g., a targetnucleic acid), to which a guide RNA hybridizes. All or part of the guideRNA sequence may be reverse complementary to all or part of the targetsequence. The target nucleic acid sequence may be adjacent to aprotospacer adjacent motif (PAM) 3′ of the target nucleic acid sequence.The PAM may promote interaction the programmable nuclease with thetarget nucleic acid. A PAM may adjacent to a DNA target nucleic acidsequence. The target nucleic acid sequence may be adjacent to aprotospacer flanking site (PFS) 3′ of the target nucleic acid sequence.The PFS may promote interaction the programmable nuclease with thetarget nucleic acid. A PFS may be adjacent to an RNA target nucleic acidsequence. One or more of the guide RNA, the PAM or PFS, or the targetnucleic acid sequence may be specifically positioned with respect to oneor more of the F1, F1c, F2, F2c, F3, F3c, LF, LFc, LB, LBc, B1, B1c, B2,B2c, B3, and/or B3c regions.

In some cases, the guide RNA is reverse complementary to a sequence ofthe target nucleic acid, which is between an F1c region and a B1 region,as in FIG. 41A. In some cases, the guide RNA is reverse complementary toa sequence of the target nucleic acid, which is between a B1c region andan F1 region.

In some cases, the guide RNA is partially reverse complementary to asequence of the target nucleic acid, which is between an F1c region anda B1 region, as in FIG. 41B. In some cases, the guide RNA is partiallyreverse complementary to a sequence of the target nucleic acid, which isbetween a B1c region and an F1 region. For example, the target nucleicacid comprises a sequence between an F1c region and a B1 region or a B1cregion and an F1 region that is reverse complementary to at least 60% ofa guide nucleic acid. In another example, the target nucleic acidcomprises a sequence between an F1c region and a B1 region that isreverse complementary to at least 10%, at least 11%, at least 12%, atleast 13%, at least 14%, at least 15%, at least 16%, at least 17%, atleast 18%, at least 19%, at least 20%, at least 21%, at least 22%, atleast 23%, at least 24%, at least 25%, at least 26%, at least 27%, atleast 28%, at least 29%, at least 30%, at least 31%, at least 32%, atleast 33%, at least 34%, at least 35%, at least 36%, at least 37%, atleast 38%, at least 39%, at least 40%, at least 41%, at least 42%, atleast 43%, at least 44%, at least 45%, at least 46%, at least 47%, atleast 48%, at least 49%, at least 50%, at least 51%, at least 52%, atleast 53%, at least 54%, at least 55%, at least 56%, at least 57%, atleast 58%, at least 59%, at least 60%, at least 61%, at least 62%, atleast 63%, at least 64%, at least 65%, at least 66%, at least 67%, atleast 68%, at least 69%, at least 70%, at least 71%, at least 72%, atleast 73%, at least 74%, at least 75%, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 100%, from 5% to 100%, from 5% to 10%,from 10% to 15%, from 15% to 20%, from 20% to 25%, from 25% to 30%, from30% to 35%, from 35% to 40%, from 40% to 45%, from 45% to 50%, from 50%to 55%, from 55% to 60%, from 60% to 65%, from 65% to 70%, from 70% to75%, from 75% to 80%, from 80% to 85%, from 85% to 90%, from 90% to 95%,or from 95% to 100% of a guide nucleic acid. In this arrangement, theguide RNA is not reverse complementary to the forward inner primer orthe backward inner primer shown in FIG. 40.

In some cases, the guide RNA is reverse complementary to no more than50%, no more than 40%, no more than 35%, no more than 30%, no more than25%, no more than 20%, no more than 15%, no more than 10%, or no morethan 5% of the forward inner primer, the backward inner primer, or acombination thereof. the sequence between the F1c region and the B1region or the sequence between the B1c region and the F1 region is atleast 50%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 92%, at least 95%, at least 99%, or 100% reversecomplementary to the guide nucleic acid sequence. In some cases, theguide nucleic acid has a sequence reverse complementary to no more than50%, no more than 40%, no more than 35%, no more than 30%, no more than25%, no more than 20%, no more than 15%, no more than 10%, or no morethan 5% of the forward inner primer, the backward inner primer, theforward outer primer, the backward outer primer, or any combinationthereof. In some cases, the guide nucleic acid sequence has a sequencereverse complementary to no more than 50%, no more than 40%, no morethan 35%, no more than 30%, no more than 25%, no more than 20%, no morethan 15%, no more than 10%, or no more than 5% of a sequence of an F3cregion, an F2c region, the F1c region, the B1c region, an B2c region, anB3c region, or any combination thereof. In some embodiments, a sequenceof the primer and a sequence of the guide nucleic acid overlap by 50% orless. In some embodiments, a sequence of the primer and a sequence ofthe guide nucleic acid do not overlap. In some embodiments, the primeris a forward primer, a reverse primer, a forward inner primer, or areverse inner primer.

In some cases, the region corresponding to the guide RNA sequence doesnot overlap or hybridize to any of the primers and may further notoverlap with or hybridize to any of the regions shown in FIG. 40-FIG. 42and FIG. 50-FIG. 51.

In some cases, all or a portion of the guide nucleic acid is reversecomplementary to a sequence of the target nucleic acid in a loop region.For example, all or a portion of the sequence of the target nucleic acidthat hybridizes to the gRNA may be located between the B1 and B2regions, as shown in FIG. 41C. In another example, all or a portion ofthe sequence of the target nucleic acid that hybridizes to the gRNA maybe located between the F2c and F1c regions, as shown in FIG. 41D. Insome cases, all or a portion of the sequence of the target nucleic acidthat hybridizes to the gRNA may be located between the F1 and F2regions. In some cases, all or a portion of the sequence of the targetnucleic acid that hybridizes to the gRNA may be located between the B2cand B1c regions.

In some cases, a LAMP primer set may be designed using a commerciallyavailable primer design software. A LAMP primer set may be designed foruse in combination with a DETECR reaction, a reverse transcriptionreaction, or both. In some cases, a LAMP primer set may be designedusing distributed ledger technology (DLT), artificial intelligence (AI),extended reality (XR) and quantum computing, commonly called “DARQ.” Insome cases, a LAMP primer set may be designed using quenching ofunincorporated amplification signal reporters (QUASR) (Ball et al., AnalChem. 2016 Apr. 5; 88(7):3562-8. doi: 10.1021/acs.analchem.5b04054. Epub2016 Mar. 24). These methods of designing a set of LAMP primers areprovided by way of example only; other methods of designing a set ofLAMP primers may be readily apparent to one skilled in the art and maybe employed in any of the compositions, kits and methods describedherein. Exemplary sets of LAMP primers for use in a combined RT-LAMPDETECTR reaction or LAMP-DETECTR to detect the presence of a nucleicacid sequence corresponding to a respiratory syncytial virus (RSV), aninfluenza A virus (IAV), an influenza B virus (IAV), or a HERC2 SNP areprovided in TABLE 5.

TABLE 5 Exemplary LAMP Primers SEQ ID NO: Primer Name Primer SetSequence SEQ ID NO: 138 F3 RSV-A- #1 TGGAACAAGTTGTGGAGG set13SEQ ID NO: 139 B3 RSV-A- #1 TGCAGCATCATATAGATCTTGA set13 SEQ ID NO: 140FIP RSV-A- #1 TAGTGATGCTTTTGGGTTGTTCAA set13 TTGTATGAGTATGCTCAAAAATTG GSEQ ID NO: 141 BIP RSV-A- #1 GTGTAGTATTGGGCAATGCTGCTC set13CTTGGTGTACCTCTGT SEQ ID NO: 142 LF RSV-A- #1 TATGGTAGAATCCTGCTTCTCCset13 SEQ ID NO: 143 LB RSV-A- #1 TGGCCTAGGCATAATGGGAGA set13SEQ ID NO: 144 F3 RSV-A- #2 AACAAGTTGTGGAGGTGTA set14 SEQ ID NO: 145B3 RSV-A- #2 CCATTTTCTTTGAGTTGTTCAG set14 SEQ ID NO: 146 FIP RSV-A- #2TAGTGATGCTTTTGGGTTGTTCAA set14 GAGTATGCTCAAAAATTGGGTG SEQ ID NO: 147BIP RSV-A- #2 GTATTGGGCAATGCTGCTGGCAT set14 ATAGATCTTGATTCCTTGGTGSEQ ID NO: 148 LF RSV-A- #2 ATATGGTAGAATCCTGCTTCTC set14 SEQ ID NO: 149LB RSV-A- #2 CCTAGGCATAATGGGAGAATAC set14 SEQ ID NO: 144 F3 RSV-A- #3AACAAGTTGTGGAGGTGTA set15 SEQ ID NO: 145 B3 RSV-A- #3CCATTTTCTTTGAGTTGTTCAG set15 SEQ ID NO: 150 FIP RSV-A- #3ATAGTGATGCTTTTGGGTTGTTCA set15 AGTATGCTCAAAAATTGGGTG SEQ ID NO: 151BIP RSV-A- #3 GCTGCTGGCCTAGGCATAATGCA set15 TCATATAGATCTTGATTCCTTSEQ ID NO: 406 LF RSV-A- #3 TATATGGTAGAATCCTGCTTCTC  set15SEQ ID NO: 152 LB RSV-A- #3 GGGAGAATACAGAGGTACAC set15 SEQ ID NO: 153F3 RSV-A- #4 GGGTCTTAGCAAAATCAGTT set16 SEQ ID NO: 139 B3 RSV-A- #4TGCAGCATCATATAGATCTTGA set16 SEQ ID NO: 154 FIP RSV-A- #4GAATCCTGCTTCTCCACCCAATTG set16 ACACGCTAGTGTACAAGC SEQ ID NO: 141BIP RSV-A- #4 GTGTAGTATTGGGCAATGCTGCTC set16 CTTGGTGTACCTCTGTSEQ ID NO: 155 LF RSV-A- #4 CCTCCACAACTTGTTCCATTTCT set16 SEQ ID NO: 156LB RSV-A- #4 TGGCCTAGGCATAATGGGAG set16 SEQ ID NO: 157 F3 RSV-A- #5AAGCAGAAATGGAACAAGTT set17 SEQ ID NO: 145 B3 RSV-A- #5CCATTTTCTTTGAGTTGTTCAG set17 SEQ ID NO: 158 FIP RSV-A- #5TAGTGATGCTTTTGGGTTGTTCAG set17 TGGAGGTGTATGAGTATGC SEQ ID NO: 159BIP RSV-A- #5 GTAGTATTGGGCAATGCTGCTGAT set17 ATAGATCTTGATTCCTTGGTGSEQ ID NO: 160 LF RSV-A- #5 TGCTTCTCCACCCAATTTTTGA set17 SEQ ID NO: 161LB RSV-A- #5 GCCTAGGCATAATGGGAGAATAC set17 SEQ ID NO: 153 F3 RSV-A- #6GGGTCTTAGCAAAATCAGTT set18 SEQ ID NO: 139 B3 RSV-A- #6TGCAGCATCATATAGATCTTGA set18 SEQ ID NO: 162 FIP RSV-A- #6GAATCCTGCTTCTCCACCCAGACA set18 CGCTAGTGTACAAGC SEQ ID NO: 141 BIP RSV-A-#6 GTGTAGTATTGGGCAATGCTGCTC set18 CTTGGTGTACCTCTGT SEQ ID NO: 155LF RSV-A- #6 CCTCCACAACTTGTTCCATTTCT set18 SEQ ID NO: 156 LB RSV-A- #6TGGCCTAGGCATAATGGGAG set18 SEQ ID NO: 163 F3 RSV-A- #7TACACAGCTGCTGTTCAA set19 SEQ ID NO: 164 B3 RSV-A- #7 GGTAAATTTGCTGGGCATTset19 SEQ ID NO: 165 FIP RSV-A- #7 TTGGAACATGGGCACCCATAAAT set19GTCCTAGAAAAAGACGATG SEQ ID NO: 166 BIP RSV-A- #7 CTAGTGAAACAAATATCCACACCset19 CAGCACTGCACTTCTTGAGTT SEQ ID NO: 167 LF RSV-A- #7TTGTAAGTGATGCAGGAT set19 SEQ ID NO: 168 LB RSV-A- #7AGGGACCCTCATTAAGAGTCATG set19 SEQ ID NO: 169 F3 RSV-A- #8ATACACAGCTGCTGTTCA set20 SEQ ID NO: 164 B3 RSV-A- #8 GGTAAATTTGCTGGGCATTset20 SEQ ID NO: 170 FIP RSV-A- #8 TCTGCTGGCATGGATGATTGAATG set20TCCTAGAAAAAGACGATG SEQ ID NO: 166 BIP RSV-A- #8 CTAGTGAAACAAATATCCACACCset20 CAGCACTGCACTTCTTGAGTT SEQ ID NO: 171 LF RSV-A- #8CCCATATTGTAAGTGATGCAGGA set20 T SEQ ID NO: 172 LB RSV-A- #8AGGGACCCTCATTAAGAGTCAT set20 SEQ ID NO: 169 F3 RSV-A- #9ATACACAGCTGCTGTTCA set21 SEQ ID NO: 173 B3 RSV-A- #9 TGGTAAATTTGCTGGGCATset21 SEQ ID NO: 170 FIP RSV-A- #9 TCTGCTGGCATGGATGATTGAATG set21TCCTAGAAAAAGACGATG SEQ ID NO: 174 BIP RSV-A- #9 TGAAACAAATATCCACACCCAAGset21 GGCACTGCACTTCTTGAGTT SEQ ID NO: 175 LF RSV-A- #9CCATATTGTAAGTGATGCAGGAT set21 SEQ ID NO: 176 LB RSV-A- #9GACCCTCATTAAGAGTCATGAT set21 SEQ ID NO: 177 F3 RSV-A- #10 AACATACGTGAACAAACTTCA set22 SEQ ID NO: 178 B3 RSV-A- #10 GCACATATGGTAAATTTGCTGG set22 SEQ ID NO: 179 FIP RSV-A- #10 ACCCATATTGTAAGTGATGCAGG set22 ATAGGGCTCCACATACACAG SEQ ID NO: 180BIP RSV-A- #10  CTAGTGAAACAAATATCCACACC set22 CAAGCACTGCACTTCTTGAGSEQ ID NO: 181 LF RSV-A- #10  TTTCTAGGACATTGTATTGAACAG set22 CSEQ ID NO: 182 LB RSV-A- #10  GGGACCCTCATTAAGAGTCATG set22SEQ ID NO: 183 IAV-MP-F3 #1 GACTTGAAGATGTCTTTGC SEQ ID NO: 184 IAV-MP B3#1 TGTTGTTTGGGTCCCCATT SEQ ID NO: 185 IAV-MP-FIP #1TTAGTCAGAGGTGACAGGATTGC AGATCTTGAGGCTCTC SEQ ID NO: 186 IAV-MP-BIP #1TTGTGTTCACGCTCACCGTGTTTG GACAAAGCGTCTACG SEQ ID NO: 187 IAV-MP FL #1GTCTTGTCTTTAGCCA SEQ ID NO: 188 IAV-MP BL #1 CAGTGAGCGAGGACTGSEQ ID NO: 189 IAV F3 v2 #2 ACCGAGGTCGAAACGT SEQ ID NO: 190 IAV B3 v2 #2GGTCCCCATTCCCATTG SEQ ID NO: 191 IAV FIP v2 #2 CAAAGACATCTTCAAGTCTCTGCGTTTTTTCTCTCTATCGTCCCGTCA SEQ ID NO: 192 IAV BIP v2 #2AATGGCTAAAGACAAGACCAATC CTTTTTTGTCTACGCTGCAGTCC SEQ ID NO: 193 IAV LF v2#2 CGATCTCGGCTTTGAGGG SEQ ID NO: 194 IAV LB v2 #2 TCACCGTGCCCAGTGAGSEQ ID NO: 195 IAV F3 v3 #3 CGAAAGCAGGTAGATATTGAAAG SEQ ID NO: 196IAV B3 v3 #3 TCTACGCTGCAGTCCTC SEQ ID NO: 197 IAV FIP v3 #3TCAAGTCTCTGCGCGATCTCTTTT TTGAGTCTTCTAACCGAGGT SEQ ID NO: 198 IAV BIP v3#3 AGATGTCTTTGCAGGGAAAAACA CTTTTTTCACAAATCCTAAAATCC CCTTAGSEQ ID NO: 199 IAV LF v3 #3 GACGATAGAGAGAACGTACGTTT C SEQ ID NO: 200IAV LB v3 #3 AAGACCAATCCTGTCACCTCT SEQ ID NO: 201 IAV-set4-F3 #4GCGAAAGCAGGTAGATATTGA SEQ ID NO: 202 IAV-set4-B3 #4 CATTCCCATTGAGGGCATTSEQ ID NO: 203 IAV-set4-FIP #4 CTTCAAGTCTCTGCGCGATCTATGAGTCTTCTAACCGAGGT SEQ ID NO: 204 IAV-set4-BIP #4 TTGAGGCTCTCATGGAATGGCAGCGTGAACACAAATCCTAA SEQ ID NO: 205 IAV-set4-LF #4 TGACGGGACGATAGAGAGAASEQ ID NO: 206 IAV-set4-LB #4 ACAAGACCAATCCTGTCACC SEQ ID NO: 201IAV-set5-F3 #5 GCGAAAGCAGGTAGATATTGA SEQ ID NO: 202 IAV-set5-B3 #5CATTCCCATTGAGGGCATT SEQ ID NO: 207  IAV-set5-FIP #5TTCAAGTCTCTGCGCGATCTCATG AGTCTTCTAACCGAGGT SEQ ID NO: 204 IAV-set5-BIP#5 TTGAGGCTCTCATGGAATGGCAG CGTGAACACAAATCCTAA SEQ ID NO: 205 IAV-set5-LF#5 TGACGGGACGATAGAGAGAA SEQ ID NO: 206 IAV-set5-LB #5ACAAGACCAATCCTGTCACC SEQ ID NO: 201 IAV-set6-F3 #6 GCGAAAGCAGGTAGATATTGASEQ ID NO: 208 IAV-set6-B3 #6 TTGGACAAAGCGTCTACG SEQ ID NO: 203IAV-set6-FIP #6 CTTCAAGTCTCTGCGCGATCTATG AGTCTTCTAACCGAGGTSEQ ID NO: 204 IAV-set6-BIP #6 TTGAGGCTCTCATGGAATGGCAGCGTGAACACAAATCCTAA SEQ ID NO: 205 IAV-set6-LF #6 TGACGGGACGATAGAGAGAASEQ ID NO: 206 IAV-set6-LB #6 ACAAGACCAATCCTGTCACC SEQ ID NO: 201IAV-set7-F3 #7 GCGAAAGCAGGTAGATATTGA SEQ ID NO: 202 IAV-set7-B3 #7CATTCCCATTGAGGGCATT SEQ ID NO: 209 IAV-set7-FIP #7AAGTCTCTGCGCGATCTCGATGA GTCTTCTAACCGAGGT SEQ ID NO: 204 IAV-set7-BIP #7TTGAGGCTCTCATGGAATGGCAG CGTGAACACAAATCCTAA SEQ ID NO: 205 IAV-set7-LF #7TGACGGGACGATAGAGAGAA SEQ ID NO: 206 IAV-set7-LB #7 ACAAGACCAATCCTGTCACCSEQ ID NO: 210 IAV-set8-F3 #8 TCTTCTAACCGAGGTCGAA SEQ ID NO: 211IAV-set8-B3 #8 CTGCTCTGTCCATGTTGTT SEQ ID NO: 212 IAV-set8-FIP #8TCAGAGGTGACAGGATTGGTCTG AAGATGTCTTTGCAGGGAA SEQ ID NO: 213 IAV-set8-BIP#8 TTGTGTTCACGCTCACCGTCATTC CCATTGAGGGCATT SEQ ID NO: 214 IAV-set8-LF #8ATTCCATGAGAGCCTCAAGATC SEQ ID NO: 215 IAV-set8-LB #8 GAGGACTGCAGCGTAGACSEQ ID NO: 216 IAV-set9-F3 #9 TTCTCTCTATCGTCCCGTC SEQ ID NO: 211IAV-set9-B3 #9 CTGCTCTGTCCATGTTGTT SEQ ID NO: 217 IAV-set9-FIP #9CCCTTAGTCAGAGGTGACAGGAA CACAGATCTTGAGGCTCT SEQ ID NO: 213 IAV-set9-BIP#9 TTGTGTTCACGCTCACCGTCATTC CCATTGAGGGCATT SEQ ID NO: 218 IAV-set9-LF #9GGTCTTGTCTTTAGCCATTCCA SEQ ID NO: 215 IAV-set9-LB #9 GAGGACTGCAGCGTAGACSEQ ID NO: 219 IAV-set10-F3 #10  GTCTTCTAACCGAGGTCGA SEQ ID NO: 211IAV-set10-B3 #10  CTGCTCTGTCCATGTTGTT SEQ ID NO: 220 IAV-set10-FIP #10 GAGGTGACAGGATTGGTCTTGTT GAAGATGTCTTTGCAGGG SEQ ID NO: 213 IAV-set10-BIP#10  TTGTGTTCACGCTCACCGTCATTC CCATTGAGGGCATT SEQ ID NO: 214 IAV-set10-LF#10  ATTCCATGAGAGCCTCAAGATC SEQ ID NO: 215 IAV-set10-LB #10 GAGGACTGCAGCGTAGAC SEQ ID NO: 221 IAV-set11-F3 #11  AAGAAGACAAGAGATATGGCSEQ ID NO: 222 IAV-set11-B3 #11  CAATTCGACACTAATTGATGGC SEQ ID NO: 223IAV-set11-FIP #11  GTCTCCTTGCCCAATTAGCAAGCA TCAATGAACTGAGCASEQ ID NO: 224 IAV-set11-BIP #11  GTGGTGTTGGTAATGAAACGAAGCTGTCTGGCTGTCAGTA SEQ ID NO: 225 IAV-set11-LF #11  ACATTAGCCTTCTCTCCTTTSEQ ID NO: 226 IAV-set11-LB #11  AACGGGACTCTAGCATACT SEQ ID NO: 227M605 F3 IBV IBV AGGGACATGAACAACAAAGA LAMP SEQ ID NO: 228 M606 B3 IBV IBVCAAGTTTAGCAACAAGCCT LAMP SEQ ID NO: 229 M607 FIP IBV IBVTCAGGGACAATACATTACGCATA LAMP TCGATAAAGGAGGAAGTAAACAC TCA SEQ ID NO: 230M608 BIP IBV IBV TAAACGGAACATTCCTCAAACAC LAMP CACTCTGGTCATATGCATTCSEQ ID NO: 231 M609 LF IBV IBV TCAAACGGAACTTCCCTTCTTTC LAMPSEQ ID NO: 232 M610 LB IBV IBV GGATACAAGTCCTTATCAACTCTG LAMP CSEQ ID NO: 233 M948 F3 HERC2 CTTGTAATCAACATCAGGGTAA HERC2 set3SEQ ID NO: 234 M949 B3 HERC2 AGAAACGACAAGTAGACCATT HERC2 set3SEQ ID NO: 235 M950 FIP HERC2 CGCCTCTTGGATCAGACACATGTG HERC2 set3TTAATACAAAGGTACAGGA SEQ ID NO: 236 M951 BIP HERC2CACGCTATCATCATCAGGGGCTG HERC2 set3 CTTCAAGTGTATATAAACTCAC SEQ ID NO: 237M952 LF HERC2 GAGAGCCATGAAGAACAAATTCT HERC2 set3 SEQ ID NO: 238 M953 LBHERC2 CGAGGCTTCTCTTTGTTTTTAAT HERC2 set3

A set of LAMP primers may be designed to introduce a PAM sequence into atarget nucleic acid sequence that lacks a PAM sequence. The FIP primermay contain a PAM sequence that is not present in the target nucleicacid. The BIP primer may contain a PAM sequence that is not present inthe target nucleic acid. The FIP primer may contain a sequence that isreverse complementary to a PAM sequence that is not present in thetarget nucleic acid. The BIP primer may contain a sequence that isreverse complementary to a PAM sequence that is not present in thetarget nucleic acid. The PAM sequence or the sequence complementary tothe PAM sequence may be located within the FIP primer or the BIP primerat a distance in bases from the 5′ end of the primer. For example, thePAM sequence may be located 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases from 5′ end of theprimer. In some embodiments, the PAM sequence may be located from 0 to10, from 5 to 15, from 10 to 20, from 15 to 25, from 20 to 30, from 25to 35, from 30 to 40 bases from 5′ end of the primer.

A set of LAMP primers may be designed for use in combination with aDETECTR reaction to detect a single nucleotide polymorphism (SNP) in atarget nucleic acid. In some embodiments, a sequence of the targetnucleic acid comprising the SNP may be reverse complementary to all or aportion of the guide nucleic acid. For example, the SNP may bepositioned within a sequence of the target nucleic acid that is reversecomplementary to the guide RNA sequence, as illustrated in FIG. 51C. Insome cases, the sequence of the target nucleic acid sequence comprisingthe SNP does not overlap with or is not reverse complementary to theprimers or one or more of the F1, F1c, F2, F2c, F3, F3c, B1, B1c, B2,B2c, B3, B3c, LB, LBc, LF, or LFc regions shown in FIG. 51. The guidenucleic acid may be reverse complementary to a sequence of the targetnucleic acid between the F1c and B1 regions, as illustrated in FIG. 51A.The guide nucleic acid may be reverse complementary to a sequence of thetarget nucleic acid between the B1c and F1 regions. A guide nucleic acidmay be partially reverse complementary to a sequence of the targetnucleic acid between the F1c region and the B1 region, for example asillustrated in FIG. 51B. A guide nucleic acid may be partially reversecomplementary to a sequence of the target nucleic acid between the B1cregion and the F1 region. For example, the sequence of the targetnucleic acid sequence having the SNP may be reverse complementary to atleast 10%, at least 11%, at least 12%, at least 13%, at least 14%, atleast 15%, at least 16%, at least 17%, at least 18%, at least 19%, atleast 20%, at least 21%, at least 22%, at least 23%, at least 24%, atleast 25%, at least 26%, at least 27%, at least 28%, at least 29%, atleast 30%, at least 31%, at least 32%, at least 33%, at least 34%, atleast 35%, at least 36%, at least 37%, at least 38%, at least 39%, atleast 40%, at least 41%, at least 42%, at least 43%, at least 44%, atleast 45%, at least 46%, at least 47%, at least 48%, at least 49%, atleast 50%, at least 51%, at least 52%, at least 53%, at least 54%, atleast 55%, at least 56%, at least 57%, at least 58%, at least 59%, atleast 60%, at least 61%, at least 62%, at least 63%, at least 64%, atleast 65%, at least 66%, at least 67%, at least 68%, at least 69%, atleast 70%, at least 71%, at least 72%, at least 73%, at least 74%, atleast 75%, at least 76%, at least 77%, at least 78%, at least 79%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 100%, from 5% to 100%, from 5% to 10%, from 10% to 15%, from 15%to 20%, from 20% to 25%, from 25% to 30%, from 30% to 35%, from 35% to40%, from 40% to 45%, from 45% to 50%, from 50% to 55%, from 55% to 60%,from 60% to 65%, from 65% to 70%, from 70% to 75%, from 75% to 80%, from80% to 85%, from 85% to 90%, from 90% to 95%, or from 95% to 100% of theguide nucleic acid. In some cases, the guide nucleic acid does notoverlap with and/or is not reverse complementary to any of the pluralityof primers or the F1, F1c, F2, F2c, F3, F3c, B1, B1c, B2, B2c, B3, B3c,LB, LBc, LF, or LFc regions. Exemplary sets of DETECTR gRNAs for use ina combined RT-LAMP DETECTR or LAMP-DETECTR reaction to detect thepresence of a nucleic acid sequence corresponding to a respiratorysyncytial virus (RSV), an influenza A virus (IAV), an influenza B virus(IAV), or a HERC2 SNP are provided in TABLE 6.

TABLE 6 Exemplary DETECTR Guide RNAs SEQ ID NO: gRNA Name SequenceSEQ ID NO: 239 gRNA #1 (R1118) UAAUUUCUACUAAGUGUAGAUCUUAUAAAAGAACUAGCCAA SEQ ID NO: 240 gRNA #2 (R288) UAAUUUCUACUAAGUGUAGAUACUCAAUUUCCUCACUUCUC SEQ ID NO: 241 R283 UAAUUUCUACUAAGUGUAGAUUGUUCACGCUCACCGUGCCC SEQ ID NO: 242 R781 UAAUUUCUACUAAGUGUAGAUGCCAUUCCAUGAGAGCCUCA SEQ ID NO: 243 R782 UAAUUUCUACUAAGUGUAGAUGACAAAGCGUCUACGCUGCA SEQ ID NO: 244 IBV (R778) UAAUUUCUACUAAGUGUAGAUCUAACACUCUCAGGGACAAU SEQ ID NO: 245 A SNP Position 9UAAUUUCUACUAAGUGUAGAUAGCAUUAAA (R570) UGUCAAGUUCU SEQ ID NO: 246G SNP Position 9 UAAUUUCUACUAAGUGUAGAUAGCAUUAAG (R571) UGUCAAGUUCUSEQ ID NO: 247 A SNP Position 14 UAAUUUCUACUAAGUGUAGAUAUUUGAGCA (R1138)UUAAAUGUCAA SEQ ID NO: 248 G SNP Position 14UAAUUUCUACUAAGUGUAGAUAUUUGAGCA (R1139) UUAAGUGUCAA

Amplification and Detection of a Single Nucleotide Polymorphism Allele

A DETECTR reaction may be used to detect the presence of a specificsingle nucleotide polymorphism (SNP) allele in a sample. The DETECTRreaction may produce a detectable signal, as described elsewhere herein,in the presence of a target nucleic acid comprising a specific SNPallele. The DETECTR reaction may not produce a signal in the absence ofthe target nucleic acid or in the presence of a nucleic acid sequencethat does not comprise the specific SNP allele or comprises a differentSNP allele. In some cases, a DETECTR reaction may comprise a guide RNAreverse complementary to a portion of a target nucleic acid sequencecomprising a specific SNP allele. The guide RNA and the target nucleicacid comprising the specific SNP allele may bind to and activate aprogrammable nuclease, thereby producing a detectable signal asdescribed elsewhere herein. The guide RNA and a nucleic acid sequencethat does not comprise the specific SNP allele may not bind to oractivate the programmable nuclease and may not produce a detectablesignal. In some cases, a target nucleic acid sequence that may or maynot comprise a specific SNP allele may be amplified using, for example,a LAMP amplification reaction. In some cases, the LAMP amplificationreaction may be combined with a reverse transcription reaction, aDETECTR reaction, or both. For example, the LAMP reaction may be anRT-LAMP reaction, a LAMP DETECTR reaction, or an RT-LAMP DETECTRreactions.

A method of assaying for a segment of a target nucleic acid may comprisecontacting a sample comprising a population of nucleic acids, whereinthe population comprises at least one nucleic acid comprising a segmenthaving less than 100% sequence identity to the segment of the targetnucleic acid and having no less than 50% sequence identity to thesegment of the target nucleic acid to a guide nucleic acid thathybridizes to the segment of the target nucleic acid, a detector nucleicacid, and a Cas12 nuclease (e.g., SEQ ID NO: 1 or SEQ ID NO: 11) thatcleaves the detector nucleic acid upon hybridization of the guidenucleic acid to the segment of the target nucleic acid; and assaying fora signal produced by cleavage of the detector nucleic acid, wherein thesignal is at least two-fold greater when the segment of the targetnucleic acid is present in the sample than the signal when the samplelacks the segment of the target nucleic acid. In some embodiments, thesegment of the at least one nucleic acid comprises at least two basemutations compared to the segment of the target nucleic acid. In someembodiments, the segment of the at least one nucleic acid comprises fromone to ten base mutations compared to the segment of the target nucleicacid. In some embodiments, the segment of the at least one nucleic acidcomprises one base mutation compared to the segment of the targetnucleic acid. In some embodiments, the signal produced is from two-foldto 20-fold greater when the segment of the target nucleic acid ispresent in the sample than the signal when the sample lacks the segmentof the target nucleic acid. In some embodiments, the signal produced isfrom two-fold to 10-fold greater when the segment of the target nucleicacid is present in the sample than the signal when the sample lacks thesegment of the target nucleic acid. In some embodiments, the signalproduced is from five-fold to 10-fold greater when the segment of thetarget nucleic acid is present in the sample than the signal when thesample lacks the segment of the target nucleic acid. In someembodiments, the signal produced is from 2 fold to 100 fold greater whenthe segment of the target nucleic acid is present in the sample than thesignal when the sample lacks the segment of the target nucleic acid. Insome embodiments, the signal produced is from 2 fold to 5 fold greaterwhen the segment of the target nucleic acid is present in the samplethan the signal when the sample lacks the segment of the target nucleicacid. In some embodiments, the signal produced is from 5 fold to 10 foldgreater when the segment of the target nucleic acid is present in thesample than the signal when the sample lacks the segment of the targetnucleic acid. In some embodiments, the signal produced is from 10 foldto 15 fold greater when the segment of the target nucleic acid ispresent in the sample than the signal when the sample lacks the segmentof the target nucleic acid. In some embodiments, the signal produced isfrom 15 fold to 20 fold greater when the segment of the target nucleicacid is present in the sample than the signal when the sample lacks thesegment of the target nucleic acid. In some embodiments, the signalproduced is from 20 fold to 25 fold greater when the segment of thetarget nucleic acid is present in the sample than the signal when thesample lacks the segment of the target nucleic acid. In someembodiments, the signal produced is from 25 fold to 30 fold greater whenthe segment of the target nucleic acid is present in the sample than thesignal when the sample lacks the segment of the target nucleic acid. Insome embodiments, the signal produced is from 30 fold to 35 fold greaterwhen the segment of the target nucleic acid is present in the samplethan the signal when the sample lacks the segment of the target nucleicacid. In some embodiments, the signal produced is from 35 fold to 40fold greater when the segment of the target nucleic acid is present inthe sample than the signal when the sample lacks the segment of thetarget nucleic acid. In some embodiments, the signal produced is from 40fold to 45 fold greater when the segment of the target nucleic acid ispresent in the sample than the signal when the sample lacks the segmentof the target nucleic acid. In some embodiments, the signal produced isfrom 45 fold to 50 fold greater when the segment of the target nucleicacid is present in the sample than the signal when the sample lacks thesegment of the target nucleic acid. In some embodiments, the signalproduced is from 50 fold to 60 fold greater when the segment of thetarget nucleic acid is present in the sample than the signal when thesample lacks the segment of the target nucleic acid. In someembodiments, the signal produced is from 60 fold to 70 fold greater whenthe segment of the target nucleic acid is present in the sample than thesignal when the sample lacks the segment of the target nucleic acid. Insome embodiments, the signal produced is from 70 fold to 80 fold greaterwhen the segment of the target nucleic acid is present in the samplethan the signal when the sample lacks the segment of the target nucleicacid. In some embodiments, the signal produced is from 80 fold to 90fold greater when the segment of the target nucleic acid is present inthe sample than the signal when the sample lacks the segment of thetarget nucleic acid. In some embodiments, the signal produced is from 90fold to 100 fold greater when the segment of the target nucleic acid ispresent in the sample than the signal when the sample lacks the segmentof the target nucleic acid. In some embodiments, the signal produced isfrom 100 fold to 200 fold greater when the segment of the target nucleicacid is present in the sample than the signal when the sample lacks thesegment of the target nucleic acid. In some embodiments, the signalproduced is from 2 fold to 10 fold greater when the segment of thetarget nucleic acid is present in the sample than the signal when thesample lacks the segment of the target nucleic acid. In someembodiments, the signal produced is from 20 fold to 40 fold greater whenthe segment of the target nucleic acid is present in the sample than thesignal when the sample lacks the segment of the target nucleic acid. Insome embodiments, the signal produced is from 2 fold to 50 fold greaterwhen the segment of the target nucleic acid is present in the samplethan the signal when the sample lacks the segment of the target nucleicacid. In some embodiments, the signal produced is from 1.5 fold to 100fold greater when the segment of the target nucleic acid is present inthe sample than the signal when the sample lacks the segment of thetarget nucleic acid. The guide may be reverse complementary to thesegment of the target nucleic acid. In some embodiments, the guidenucleic acid and the second guide nucleic acid lack syntheticmismatches. A synthetic mismatch may be an additional mismatch between atarget nucleic acid and a guide nucleic acid introduced into the guidenucleic acid to improve the single-base distinction capabilities of aprogrammable nuclease.

In some embodiments, the DETECTR reaction may be used to detect thepresence of a specific SNP allele in a sample, wherein the SNP islocated in a target nucleic acid sequence that lacks a PAM sequence. Forexample, the DETECTR reaction, wherein the target nucleic acid segmentlacks a PAM sequence, comprises LAMP amplifying the target nucleic acidsegment using a forward inner primer (FIP) or a backward inner primer(BIP) having a region that is reverse complementary to the targetnucleic acid segment and a region that has a PAM sequence reversecomplement, thereby generating a PAM target nucleic acid having a PAMsequence adjacent to target sequence of an amplification product;contacting the PAM target nucleic acid to PAM-dependent sequencespecific nuclease complex comprising a guide nucleic acid and aprogrammable nuclease that exhibits sequence independent cleavage uponforming a complex comprising the segment of the guide nucleic acidbinding to the segment of the PAM target nucleic acid; and assaying forcleavage of at least one nucleic acid of the reporter of a population ofnucleic acids of the reporters, wherein the cleavage indicates apresence of the target nucleic acid in the sample and wherein theabsence of the cleavage indicates an absence of the target nucleic acidin the sample. The detection of the signal can indicate the presence ofthe target nucleic acid. Sometimes, the target nucleic acid comprises amutation. Often, the mutation is a single nucleotide mutation.

The SNP may be positioned at a distance from a PAM sequence. The PAMsequence may be a native PAM sequence, or the PAM sequence may be agenerated PAM sequence. The PAM sequence may be generated byamplification. In some embodiments, the SNP may be positioned 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or40 bases from the PAM sequence. In some embodiments, the SNP may bepositioned from 1 to 10, from 5 to 15, from 10 to 20, from 15 to 25,from 20 to 30, from 25 to 35, from 30 to 40 bases from the PAM sequence.The SNP may be positioned on the forward strand. The SNP may bepositioned on the reverse strand.

A guide nucleic acid may be specific for an SNP allele. For example, aguide nucleic acid may increase the trans cleavage activity of aprogrammable nuclease more when contacted to a target nucleic acidcomprising a specific SNP allele than when contacted to a target nucleicacid comprising a different SNP allele. In some embodiments, the guidenucleic acid may increase the trans cleavage activity of a programmablenuclease more when contacted to a target nucleic acid comprising an Anucleic acid at a SNP than when contacted to a target nucleic acidcomprising a T, a C, or a G nucleic acid at the SNP. In someembodiments, the guide nucleic acid may increase the trans cleavageactivity of a programmable nuclease more when contacted to a targetnucleic acid comprising a T nucleic acid at a SNP than when contacted toa target nucleic acid comprising an A, a C, or a G nucleic acid at theSNP. In some embodiments, the guide nucleic acid may increase the transcleavage activity of a programmable nuclease more when contacted to atarget nucleic acid comprising a C nucleic acid at a SNP than whencontacted to a target nucleic acid comprising an A, a T, or a G nucleicacid at the SNP. In some embodiments, the guide nucleic acid mayincrease the trans cleavage activity of a programmable nuclease morewhen contacted to a target nucleic acid comprising a G nucleic acid at aSNP than when contacted to a target nucleic acid comprising an A, a C,or a T nucleic acid at the SNP. In some embodiments, the guide nucleicacid may be specific for a first SNP allele at a first SNP and a secondSNP allele at a second SNP site. The programmable nuclease may be aCas12, a Cas13, or a Cas14.

A DETECTR reaction, as described elsewhere herein, may produce adetectable signal specifically in the presence of a target nucleic acidsequence comprising a specific SNP allele. For example, the DETECTRreaction may produce a detectable signal in the presence of a targetnucleic acid comprising a G nucleic acid at a location of a SNP but notin the presence of a nucleic acid comprising a C, a T, or an A nucleicacid at the location of the SNP. The DETECTR reaction may produce adetectable signal in the presence of a target nucleic acid comprising aT nucleic acid at a location of a SNP but not in the presence of anucleic acid comprising a G, a C, or an A nucleic acid at the locationof the SNP. The DETECTR reaction may produce a detectable signal in thepresence of a target nucleic acid comprising a C nucleic acid at alocation of a SNP but not in the presence of a nucleic acid comprising aG, a T, or an A nucleic acid at the location of the SNP. The DETECTRreaction may produce a detectable signal in the presence of a targetnucleic acid comprising an A nucleic acid at a location of a SNP but notin the presence of a nucleic acid comprising a G, a T, or a C nucleicacid at the location of the SNP. In addition to the DETECTR reaction,the target nucleic acid having the SNP may be concurrently,sequentially, concurrently together in a sample, or sequentiallytogether in a sample be carried out alongside LAMP or RT-LAMP. Forexample, the reactions can comprise LAMP and DETECTR reactions, orRT-LAMP and DETECTR reactions. Performing a DETECTR reaction incombination with a LAMP reaction may result in an increased detectablesignal as compared to the DETECTR reaction in the absence of the LAMPreaction.

In some cases, the detectable signal produced in the DETECTR reactionmay be higher in the presence of a target nucleic acid comprising aspecific SNP allele than in the presence of a nucleic acid that does notcomprise the specific SNP allele. In some cases, the DETECTR reactionmay produce a detectable signal that is at least 1-fold, at least2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least50-fold, at least 100-fold, at least 200-fold, at least 300-fold, atlast 400-fold, at least 500-fold, at least 1000-fold, at least2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold,at least 6000-fold, at least 7000-fold, at least 8000-fold, at least9000-fold, at least 10000-fold, at least 50000-fold, at least100000-fold, at least 500000-fold, or at least 1000000-fold greater inthe presence of a target nucleic acid comprising a specific SNP allelethan in the presence of a nucleic acid that does not comprise thespecific SNP allele. In some cases, the DETECTR reaction may produce adetectable signal that is from 1-fold to 2-fold, from 2-fold to 3-fold,from 3-fold to 4-fold, from 4-fold to 5-fold, from 5-fold to 10-fold,from 10-fold to 20-fold, from 20-fold to 30-fold, from 30-fold to40-fold, from 40-fold to 50-fold, from 50-fold to 100-fold, from100-fold to 500-fold, from 500-fold to 1000-fold, from 1000-fold to10,000-fold, from 10,000-fold to 100,000-fold, or from 100,000-fold to1,000,000-fold greater in the presence of a target nucleic acidcomprising a specific SNP allele than in the presence of a nucleic acidthat does not comprise the specific SNP allele.

A DETECTR reaction may be used to detect the presence of a SNP alleleassociated with a disease or a condition in a nucleic acid sample. TheDETECTR reaction may be used to detect the presence of a SNP alleleassociated with an increased likelihood of developing a disease or acondition in a nucleic acid sample. The DETECTR reaction may be used todetect the presence of a SNP allele associated with a phenotype in anucleic acid sample. For example, a DETECTR reaction may be used todetect a SNP allele associated with a disease such as phenylketonuria(PKU), cystic fibrosis, sickle-cell anemia, albinism, Huntington'sdisease, myotonic dystrophy type 1, hypercholesterolemia,neurofibromatosis, polycystic kidney disease, hemophilia, musculardystrophy, hypophosphatemic rickets, Rett's syndrome, or spermatogenicfailure. A SNP allele associated with a disease may be in a gene such asphenylalanine hydroxylase (PAH) gene, cystic fibrosis transmembraneconductance regulator (CFTR) gene, a β-globin gene, a Huntingtin gene, adystrophin (DMD) gene, an apolipoprotein B (APOB) gene, a low-densitylipoprotein receptor (LDLR) gene, a low-density lipoprotein receptoradaptor protein 1 (LDLRAP1) gene, a proprotein convertasesubtilisin/kexin type 9 (PCSK9) gene, a neurofibromin (NF1) gene, a PKD1gene, an PKD2 gene, a coagulation factor VIII (F8) gene, a coagulationfactor IX (F9) gene, a myotonic dystrophy protein kinase (DMPK) gene, aphosphate regulating endopeptidase homolog X-linked (PHEX) gene, or amethyl CpG binding protein 2 (MECP) gene. A DETECTR reaction may be usedto detect a SNP allele associated with an increased risk of cancer, forexample bladder cancer, brain cancer, breast cancer, cervical cancer,colon cancer, colorectal cancer, gallbladder cancer, stomach cancer,leukemia, liver cancer, lung cancer, oral cancer, esophageal cancer,ovarian cancer, pancreatic cancer, prostate cancer, skin cancer,testicular cancer, thyroid cancer, neuroblastoma, or lymphoma. A DETECTRreaction may be used to detect a SNP allele associated with an increasedrisk of a disease, for example Alzheimer's disease, Parkinson's disease,amyloidosis, heterochromatosis, celiac disease, macular degeneration, orhypercholesterolemia. A DETECTR reaction may be used to detect a SNPallele associated with a phenotype, for example, eye color, hair color,height, skin color, race, alcohol flush reaction, caffeine consumption,deep sleep, genetic weight, lactose intolerance, muscle composition,saturated fat and weight, or sleep movement.

A target nucleic acid may be amplified prior to detection (e.g.,detection using a DETECTR reaction). The target nucleic acid may beamplified using any of the amplification methods or reagents describedherein. The DETECTR reaction may comprise detecting the presence of atarget nucleic acid comprising a specific SNP allele at a SNP ofinterest. In some cases, the target nucleic acid comprises a sequencevariation that is not of interest near the SNP of interest. The sequencevariation may comprise a second SNP, a heterogenous sequence, or aregion of low sequence conservation. The sequence variation may be nearthe SNP of interest. For example, the sequence variation may overlapwith an annealing region for a gRNA directed to detect a specific alleleof the SNP of interest. In some embodiments, the target nucleic acid maybe amplified prior to or concurrent with detection to reduce or removethe sequence variation that is not of interest while preserving the SNPof interest. For example, amplification to remove the sequence variationmay be performed using a primer that overlaps with or anneals to aregion of the nucleic acid comprising the sequence variation that is notof interest. The primer may not overlap or anneal to the regioncomprising the SNP of interest. In some cases, the primer overlaps witha region that corresponds to or anneals to the gRNA. Amplification usingthe primer that overlaps the sequence variation that is not of interestmay increase the homogeneity of the nucleic acid sequence at the site ofthe variation that is not of interest while maintaining theheterogeneity of the nucleic acid at the SNP of interest. For example,amplification may be used to overwrite the sequence variation that isnot of interest. In some cases, amplification to increase thehomogeneity of the nucleic acid sequence may be used improvespecies-level detection of a target nucleic acid wherein the gRNA istarget to a region of low or imperfect sequence conservation.

Detection/Visualization Devices

A number of detection or visualization devices and methods areconsistent with the methods, compositions, reagents, enzymes, and kitsdisclosed herein for assaying for a signal indicating cleavage of atleast some detector nucleic acids of a population of detector nucleicacids. The methods disclosed herein are, for example, consistent withfluidic devices for detection of a signal indicating cleavage of atleast some detector nucleic acids of a population of detector nucleicacids, wherein the fluidic device may comprise multiple pumps, valves,reservoirs, and chambers for sample preparation, amplification of atarget nucleic acid within the sample, mixing with a programmablenuclease, and detection of a signal indicating cleavage of at least somedetector nucleic acids of a population of detector nucleic acids by theprogrammable nuclease within the fluidic system itself. For example, thefluidic device may comprise an incubation and detection chamber or astand-alone detection chamber, in which a colorimetric, fluorescence,electrochemical, or electrochemiluminesence signal is generated fordetection. The detection can be analyzed using various methods.

As described herein, a target nucleic acid comprising DNA may bedetected using a DNA-activated programmable RNA nuclease and otherreagents disclosed herein. A DNA-activated programmable RNA nuclease mayalso be multiplexed as described herein. Sometimes, the signal generatedfor detection is a calorimetric, potentiometric, amperometric, optical(e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. Oftena calorimetric signal is heat produced after cleavage of the nucleicacids of a reporter. Sometimes, a calorimetric signal is heat absorbedafter cleavage of the nucleic acids of a reporter. A potentiometricsignal, for example, is electrical potential produced after cleavage ofthe nucleic acids of a reporter. An amperometric signal can be movementof electrons produced after the cleavage of a nucleic acid of areporter. Often, the signal is an optical signal, such as a colorimetricsignal or a fluorescence signal. An optical signal is, for example, alight output produced after the cleavage of the nucleic acids of areporter. Sometimes, an optical signal is a change in light absorbancebetween before and after the cleavage of nucleic acids of a reporter.Often, a piezo-electric signal is a change in mass between before andafter the cleavage of the nucleic acid of a reporter. Sometimes, thenucleic acid of a reporter is a protein-nucleic acid. Often, theprotein-nucleic acid is an enzyme-nucleic acid. Thedetection/visualization can be analyzed using various methods, asfurther described below.

The results from the detection region from a completed assay can bedetected or visualized and analyzed in various ways. In some cases, thepositive control spot and the detection spot in the detection region isvisible by eye, and the results can be read by the user. In some cases,the positive control spot and the detection spot in the detection regionis visualized by an imaging device. Often, the imaging device is adigital camera, such a digital camera on a mobile device. The mobiledevice may have a software program or a mobile application that cancapture an image of the support medium, identify the assay beingperformed, detect the detection region and the detection spot, provideimage properties of the detection spot, analyze the image properties ofthe detection spot, and provide a result. Alternatively, or incombination, the imaging device can capture fluorescence, ultraviolet(UV), infrared (IR), or visible wavelength signals. The imaging devicemay have an excitation source to provide the excitation energy andcaptures the emitted signals. In some cases, the excitation source canbe a camera flash and optionally a filter. In some cases, the imagingdevice is used together with an imaging box that is placed over thesupport medium to create a dark room to improve imaging. The imaging boxcan be a cardboard box that the imaging device can fit into beforeimaging. In some instances, the imaging box has optical lenses, mirrors,filters, or other optical elements to aid in generating a more focusedexcitation signal or to capture a more focused emission signal. Often,the imaging box and the imaging device are small, handheld, and portableto facilitate the transport and use of the assay in remote or lowresource settings.

The assay described herein can be visualized and analyzed by a mobileapplication (app) or a software program. Using the graphic userinterface (GUI) of the app or program, an individual can take an imageof the support medium, including the detection region, barcode,reference color scale, and fiduciary markers on the housing, using acamera on a mobile device. The program or app reads the barcode oridentifiable label for the test type, locate the fiduciary marker toorient the sample, and read the detectable signals, compare against thereference color grid, and determine the presence or absence of thetarget nucleic acid, which indicates the presence of the gene, virus, orthe agent responsible for the disease, cancer, or genetic disorder. Themobile application can present the results of the test to theindividual. The mobile application can store the test results in themobile application. The mobile application can communicate with a remotedevice and transfer the data of the test results. The test results canbe viewable remotely from the remote device by another individual,including a healthcare professional. A remote user can access theresults and use the information to recommend action for treatment,intervention, clean up of an environment.

The methods for detection of a target nucleic acid described hereinfurther can comprises reagents protease treatment of the sample. Thesample can be treated with protease, such as Protease K, beforeamplification or before assaying for a detectable signal. Often, aprotease treatment is for no more than 15 minutes. Sometimes, theprotease treatment is for no more than 1, 5, 10, 15, 20, 25, 30, or moreminutes, or any value from 1 to 30 minutes. Sometimes, the proteasetreatment is from 1 to 30, from 5 to 25, from 10 to 20, or from 10 to 15minutes. Sometimes, the total time for the performing the methoddescribed herein is no greater than 3 hours, 2 hours, 1 hour, 50minutes, 40 minutes, 30 minutes, 20 minutes, or any value from 3 hoursto 20 minutes. Often, a method of nucleic acid detection from a rawsample comprises protease treating the sample for no more than 15minutes, amplifying (can also be referred to as pre-amplifying) thesample for no more than 15 minutes, subjecting the sample to aprogrammable nuclease-mediated detection, and assaying nuclease mediateddetection. The total time for performing this method, sometimes, is nogreater than 3 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30minutes, 20 minutes, or any value from 3 hours to 20 minutes. Often, theprotease treatment is Protease K. Often the amplifying is thermalcycling amplification. Sometimes the amplifying is isothermalamplification.

Fluidic Devices

Disclosed herein are various fluidic devices for assaying for a signalindicating cleavage of at least some detector nucleic acids of apopulation of detector nucleic acids. The fluidic devices describedherein can be used to monitor the signal indicating cleavage of at leastsome detector nucleic acids of a population of detector nucleic acidsthat occurs when a target nucleic acid in samples binding to aprogrammable nuclease complexed with a guide nucleic acid, therebyallowing initiating cleavage of detector nucleic acids that produce asignal upon cleavage. All samples and reagents disclosed herein arecompatible for use with a fluidic device disclosed below. Anyprogrammable nuclease, such as any Cas nuclease described herein, arecompatible for use with a fluidic device disclosed below. Supportmediums and housing disclosed herein are also compatible for use inconjunction with the fluidic devices disclosed below. Multiplexingdetection, as described throughout the present disclosure, can becarried out within the fluidic devices disclosed herein. Compositionsand methods for detection and visualization disclosed herein are alsocompatible for use within the below described fluidic systems.

A workflow of a method for assaying a target nucleic acid in a samplewithin a fluidic device can include sample preparation, nucleic acidamplification, incubation with a programmable nuclease, and/or detection(readout). For example, a step 1 is sample preparation, a step 2 isnucleic acid amplification, a step 3 is programmable nucleaseincubation, and a step 4 is detection (readout). In some embodiments,amplification comprises producing a PAM target nucleic acid. Sometimes,steps 1 and 2 are optional. Steps 3 and 4 can occur concurrently, ifincubation and detection of programmable nuclease activity are withinthe same chamber. Sample preparation and amplification can be carriedout within a fluidic device described herein or, alternatively, can becarried out prior to introduction into the fluidic device. As mentionedabove, sample preparation of any nucleic acid amplification areoptional, and can be excluded. In further cases, programmable nucleasereaction incubation and detection (readout) can be performedsequentially (one after another) or concurrently (at the same time). Insome embodiments, sample preparation and/or amplification can beperformed within a first fluidic device and then the sample can betransferred to a second fluidic device to carry out Steps 3 and 4 and,optionally, Step 2.

A fluidic device for sample preparation can be referred to as afiltration device. In some embodiments, the filtration device for samplepreparation resembles a syringe or, comprises, similar functionalelements to a syringe. For example, a functional element of thefiltration device for sample preparation includes a narrow tip forcollection of liquid samples. Liquid samples can include blood, saliva,urine, or any other biological fluid. Liquid samples can also includeliquid tissue homogenates. The tip, for collection of liquid samples,can be manufactured from glass, metal, plastic, or other biocompatiblematerials. The tip may be replaced with a glass capillary that may serveas a metering apparatus for the amount of biological sample addeddownstream to the fluidic device. For some samples, e.g., blood, thecapillary may be the only fluidic device required for samplepreparation. Another functional element of the filtration device forsample preparation may include a channel that can carry volumes from nLto mL, containing lysis buffers compatible with the programmablenuclease reaction downstream of this process. The channel may bemanufactured from metal, plastic, or other biocompatible materials. Thechannel may be large enough to hold an entire fecal, buccal, or otherbiological sample collection swab. The filtration device may furthercontain a solution of reagents that will lyse the cells in each type ofsamples and release the nucleic acids so that they are accessible to theprogrammable nuclease. Active ingredients of the solution may bechaotropic agents, detergents, salts, and can be of high osmolality,ionic strength and pH. Chaotropic agents or chaotropes are substancesthat disrupt the three-dimensional structure in macromolecules such asproteins, DNA, or RNA. One example protocol comprises a 4 M guanidiniumisothiocyanate, 25 mM sodium citrate.2H₂O, 0.5% (w/v) sodium laurylsarcosinate, and 0.1 M β-mercaptoethanol), but numerous commercialbuffers for different cellular targets may also be used. Alkalinebuffers may also be used for cells with hard shells, particularly forenvironmental samples. Detergents such as sodium dodecyl sulphate (SDS)and cetyl trimethylammonium bromide (CTAB) may also be implemented tochemical lysis buffers. Cell lysis may also be performed by physical,mechanical, thermal or enzymatic means, in addition tochemically-induced cell lysis mentioned previously. The device mayinclude more complex architecture depending on the type of sample, suchas nanoscale barbs, nanowires, sonication capability in a separatechamber of the device, integrated laser, integrated heater, for example,a Peltier-type heater, or a thin-film planar heater, and/ormicrocapillary probes for electrical lysis. Any samples described hereincan be used in this workflow. For example samples may include liquidsamples collected from a subject being tested for a condition ofinterest. The sample preparation fluidic device can process differenttypes of biological sample: finger-prick blood, urine or swabs withfecal, cheek or other collection.

A fluidic device may be used to carry out any one of, or any combinationof, steps 2-4 discussed above (nucleic acid amplification, programmablenuclease reaction incubation, detection (readout)). An example fluidicdevice for a programmable nuclease reaction with a fluorescence orelectrochemical readout that may be used in Step 2 to Step 4 can becarried out in different iterations. For example, one variation is afluidic device that performs the programmable nuclease reactionincubation and detection (readout) steps, but not amplification. Anothervariation of a fluidic device comprises a one-chamber reaction withamplification. Another variation of the fluidic device comprises atwo-chamber reaction with amplification. Fluorescence or electrochemicalprocesses that may be used for detection of the reaction in a fluidicdevice as described above.

The chip (also referred to as fluidic device) may be manufactured from avariety of different materials. Exemplary materials that may be usedinclude plastic polymers, such as poly-methacrylate (PMMA), cyclicolefin polymer (COP), cyclic olefin copolymer (COC), polyethylene (PE),high-density polyethylene (HDPE), polypropylene (PP); glass; andsilicon. Features of the chip may be manufactured by various processes.For example, features may be (1) embossed using injection molding, (2)micro-milled or micro-engraved using computer numerical control (CNC)micromachining, or non-contact laser drilling (by means of a C02 lasersource); (3) additive manufacturing, and/or (4) photolithographicmethods.

The design may include up to three (3) input ports operated by three (3)pumps, for example. The pumps may be operated by external syringe pumpsusing low pressure or high pressure. The pumps may be passive, and/oractive (pneumatic, piezoelectric, Braille pin, electroosmotic, acoustic,gas permeation, or other).

The ports may be connected to pneumatic pressure pumps, air or gas maybe pumped into the microfluidic channels to control the injection offluids into the fluidic device. At least three reservoirs may beconnected to the device, each containing buffered solutions of: (1)sample, which may be a solution containing purified nucleic acidsprocessed in a separate fluidic device, or neat sample (blood, saliva,urine, stool, and/or sputum); (2) amplification mastermix, which variesdepending on the method used, wherein the method may include any ofloop-mediated isothermal amplification (LAMP), strand displacementamplification (SDA), recombinase polymerase amplification (RPA),helicase dependent amplification (HDA), multiple displacementamplification (MDA), rolling circle amplification (RCA), and nucleicacid sequence-based amplification (NASBA), transcription mediatedamplification (TMA), circular helicase dependent amplification (cHDA),exponential amplification reaction (EXPAR), ligase chain reaction (LCR),simple method amplifying RNA targets (SMART), single primer isothermalamplification (SPIA), hinge-initiated primer-dependent amplification ofnucleic acids (HIP), nicking enzyme amplification reaction (NEAR), orimproved multiple displacement amplification (IMDA); and (3)pre-complexed programmable nuclease mix, which includes one or moreprogrammable nuclease and guide oligonucleotides. The method of nucleicacid amplification may also be polymerase chain reaction (PCR), whichincludes cycling of the incubation temperature at different levels,hence is not defined as isothermal. Often, the reagents for nucleic acidamplification comprise a recombinase, a oligonucleotide primer, asingle-stranded DNA binding (SSB) protein, and a polymerase. Sometimes,nucleic acid amplification of the sample improves at least one ofsensitivity, specificity, or accuracy of the assay in detecting thetarget nucleic acid. In some cases, the nucleic acid amplification isperformed in a nucleic acid amplification region on the support medium.The nucleic amplification can produce a PAM target nucleic acid asdisclosed by the methods herein. Alternatively or in combination, thenucleic acid amplification is performed in a reagent chamber, and theresulting sample is applied to the support medium. Sometimes, thenucleic acid amplification is isothermal nucleic acid amplification.Complex formation of a nuclease with guides (a programmable nuclease)and reporter probes may occur off the chip. An additional port foroutput of the final reaction products is depicted at the end of thefluidic path, and is operated by a similar pump. The reactions productcan be, thus, collected for additional processing and/orcharacterization, e.g., sequencing.

The flow of liquid in this fluidic device may be controlled using up tofour (4) microvalves. These valves can be electro-kinetic microvalves,pneumatic microvalves, vacuum microvalves, capillary microvalves, pinchmicrovalves, phase-change microvalves, burst microvalves.

The flow to and from the fluidic channel from each microvalve can becontrolled by valves. The volume of liquids pumped into the ports canvary from nL to mL depending in the overall size of the device.

A fluidic device in which no amplification is needed can also be used.After addition of sample and pre-complexed programmable nuclease mix,these reagents may be mixed in a serpentine channel which then leads toa chamber where the mixture may be incubated at the required temperatureand time. The readout can be done simultaneously in the chamber.Thermoregulation in the chamber may be carried out using a thin-filmplanar heater manufactured, from e.g. Kapton, or other similarmaterials, and controlled by a proportional integral derivative (PID).

A fluidic device may also allow for addition of sample, amplificationmix, and pre-complexed programmable nuclease mix, the reagents to thenbe mixed in a serpentine channel which then leads to a chamber where themixture is incubated at the required temperature and time needed toefficient amplification, using any of the amplification methodsdescribed herein. The readout may be done simultaneously in the chamber.Thermoregulation may be achieved as previously described.

A fluidic device can allow for amplification and programmable nucleasereactions occur in separate chambers. The pre-complexed programmablenuclease mix can be pumped into the amplified mixture from a firstchamber using a pump. The liquid flow is controlled by a valve, anddirected into a serpentine mixer, and subsequently in another chamberfor incubation the required temperature, for example at 37° C. for 90minutes.

During the detection step (step 4, for example), the programmablenuclease complexed to a guide nucleic acid binds to its target nucleicacid from the amplified sample to initiate cleavage of a detectornucleic acid to generate a signal readout. In the absence of a targetnucleic acid, the programmable nuclease complexed to a guide nucleicacid does not cleave the detector nucleic acid. Detection of the signalcan be achieved by multiple methods, which can detect a signal that iscalorimetric, potentiometric, amperometric, optical (e.g., fluorescent,colorimetric, etc.), or piezo-electric, as non-limiting examples.

Support Medium

A number of support mediums are consistent with the methods disclosedherein. These support mediums are, for example, consistent with fluidicdevices disclosed herein for detection of a target nucleic acid sequencewithin the sample, wherein the fluidic device may comprise multiplepumps, valves, reservoirs, and chambers for sample preparation,amplification of a target nucleic acid sequence within the sample,mixing with a programmable nuclease, and detection of a detectablesignal arising from cleavage of detector nucleic acids by theprogrammable nuclease within the fluidic system itself. These supportmediums are compatible with the DETECTR assay methods disclosed herein.The support mediums, as described herein, are compatible with any of theprogrammable nucleases disclosed herein (e.g., a programmable nucleasewith at least 60% sequence identity to SEQ ID NO: 11) and use of saidprogrammable nuclease in a method of detecting a target nucleic acid.The support mediums, as described herein, are compatible with any of thecompositions comprising a programmable nuclease and a buffer, which hasbeen developed to improve the function of the programmable nuclease(e.g., a programmable nuclease and a buffer with low salt (about 110 mMor less) and a pH of 7 to 8) and use of said compositions in a method ofdetecting a target nucleic acid. The support mediums, as describedherein, are compatible with any of the methods disclosed hereinincluding methods of assaying for at least one base difference (e.g.,assaying for a SNP or a base mutation) in a target nucleic acidsequence, methods of assaying for a target nucleic acid that lacks a PAMby amplifying the target nucleic acid sequence to introduce a PAM, andcompositions used in introducing a PAM via amplification into the targetnucleic acid sequence. In some embodiments, amplification of the targetnucleic acid sequence within the sample comprises producing a PAM targetnucleic acid. These support mediums are compatible with the samples,reagents, and fluidic devices described herein for detection of anailment, such as a disease, cancer, or genetic disorder, or geneticinformation, such as for phenotyping, genotyping, or determiningancestry. A support medium described herein can provide a way to presentthe results from the activity between the reagents and the sample. Thesupport medium provides a medium to present the detectable signal in adetectable format. Optionally, the support medium concentrates thedetectable signal to a detection spot in a detection region to increasethe sensitivity, specificity, or accuracy of the assay. The supportmediums can present the results of the assay and indicate the presenceor absence of the disease of interest targeted by the target nucleicacid. The result on the support medium can be read by eye or using amachine. The support medium helps to stabilize the detectable signalgenerated by the cleaved detector molecule on the surface of the supportmedium. In some instances, the support medium is a lateral flow assaystrip. In some instances, the support medium is a PCR plate. The PCRplate can have 96 wells or 384 wells. The PCR plate can have a subsetnumber of wells of a 96 well plate or a 384 well plate. A subset numberof wells of a 96 well PCR plate is, for example, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 wells. Forexample, a PCR subset plate can have 4 wells wherein a well is the sizeof a well from a 96 well PCR plate (e.g., a 4 well PCR subset platewherein the wells are the size of a well from a 96 well PCR plate). Asubset number of wells of a 384 well PCR plate is, for example, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280,300, 320, 340, 360, or 380 wells. For example, a PCR subset plate canhave 20 wells wherein a well is the size of a well from a 384 well PCRplate (e.g., a 20 well PCR subset plate wherein the wells are the sizeof a well from a 384 well PCR plate). The PCR plate or PCR subset platecan be paired with a fluorescent light reader, a visible light reader,or other imaging device. Often, the imaging device is a digital camera,such a digital camera on a mobile device. The mobile device may have asoftware program or a mobile application that can capture an image ofthe PCR plate or PCR subset plate, identify the assay being performed,detect the individual wells and the sample therein, provide imageproperties of the individuals wells comprising the assayed sample,analyze the image properties of the contents of the individual wells,and provide a result.

The support medium has at least one specialized zone or region topresent the detectable signal. The regions comprise at least one of asample pad region, a nucleic acid amplification region, a conjugate padregion, a detection region, and a collection pad region. In someinstances, the regions are overlapping completely, overlappingpartially, or in series and in contact only at the edges of the regions,where the regions are in fluid communication with its adjacent regions.In some instances, the support medium has a sample pad located upstreamof the other regions; a conjugate pad region having a means forspecifically labeling the detector moiety; a detection region locateddownstream from sample pad; and at least one matrix which defines a flowpath in fluid connection with the sample pad. In some instances, thesupport medium has an extended base layer on top of which the variouszones or regions are placed. The extended base layer may provide amechanical support for the zones.

Described herein are sample pad that provide an area to apply the sampleto the support medium. The sample may be applied to the support mediumby a dropper or a pipette on top of the sample pad, by pouring ordispensing the sample on top of the sample pad region, or by dipping thesample pad into a reagent chamber holding the sample. The sample can beapplied to the sample pad prior to reaction with the reagents when thereagents are placed on the support medium or be reacted with thereagents prior to application on the sample pad. The sample pad regioncan transfer the reacted reagents and sample into the other zones of thesupport medium. Transfer of the reacted reagents and sample may be bycapillary action, diffusion, convection or active transport aided by apump. In some cases, the support medium is integrated with or overlayedby microfluidic channels to facilitate the fluid transport.

The dropper or the pipette may dispense a predetermined volume. In somecases, the predetermined volume may range from about 1 μl to about 1000μl, about 1 μl to about 500 μl, about 1 μl to about 100 μl, or about 1μl to about 50 μl. In some cases, the predetermined volume may be atleast 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 25μl, 50 μl, 75 μl, 100 μl, 250 μl, 500 μl, 750 μl, or 1000 μl. Thepredetermined volume may be no more than 5 μl, 10 μl, 25 μl, 50 μl, 75μl, 100 μl, 250 μl, 500 μl, 750 μl, or 1000 μl. The dropper or thepipette may be disposable or be single-use.

Optionally, a buffer or a fluid may also be applied to the sample pad tohelp drive the movement of the sample along the support medium. In somecases, the volume of the buffer or the fluid may range from about 1 μlto about 1000 μl, about 1 μl to about 500 μl, about 1 μl to about 100μl, or about 1 μl to about 50 μl. In some cases, the volume of thebuffer or the fluid may be at least 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl,7 μl, 8 μl, 9 μl, 10 μl, 25 μl, 50 μl, 75 μl, 100 μl, 250 μl, 500 μl,750 μl, or 1000 μl. The volume of the buffer or the fluid may be no morethan 5 μl, 10 μl, 25 μl, 50 μl, 75 μl, 100 μl, 250 μl, 500 μl, 750 μl,or 1000 μl. In some cases, the buffer or fluid may have a ratio of thesample to the buffer or fluid of at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, or 1:10.

The sample pad can be made from various materials that transfer most ofthe applied reacted reagents and samples to the subsequent regions. Thesample pad may comprise cellulose fiber filters, woven meshes, porousplastic membranes, glass fiber filters, aluminum oxide coated membranes,nitrocellulose, paper, polyester filter, or polymer-based matrices. Thematerial for the sample pad region may be hydrophilic and have lownon-specific binding. The material for the sample pad may range fromabout 50 μm to about 1000 μm, about 50 μm to about 750 μm, about 50 μmto about 500 μm, or about 100 μm to about 500 μm.

The sample pad can be treated with chemicals to improve the presentationof the reaction results on the support medium. The sample pad can betreated to enhance extraction of nucleic acid in the sample, to controlthe transport of the reacted reagents and sample or the conjugate toother regions of the support medium, or to enhance the binding of thecleaved detection moiety to the conjugate binding molecule on thesurface of the conjugate or to the capture molecule in the detectionregion. The chemicals may comprise detergents, surfactants, buffers,salts, viscosity enhancers, or polypeptides. In some instances, thechemical comprises bovine serum albumin.

Described herein are conjugate pads that provide a region on the supportmedium comprising conjugates coated on its surface by conjugate bindingmolecules that can bind to the detector moiety from the cleaved detectormolecule or to the control molecule. The conjugate pad can be made fromvarious materials that facilitate binding of the conjugate bindingmolecule to the detection moiety from cleaved detector molecule andtransfer of most of the conjugate-bound detection moiety to thesubsequent regions. The conjugate pad may comprise the same material asthe sample pad or other zones or a different material than the samplepad. The conjugate pad may comprise glass fiber filters, porous plasticmembranes, aluminum oxide coated membranes, paper, cellulose fiberfilters, woven meshes, polyester filter, or polymer-based matrices. Thematerial for the conjugate pad region may be hydrophilic, have lownon-specific binding, or have consistent fluid flow properties acrossthe conjugate pad. In some cases, the material for the conjugate pad mayrange from about 50 μm to about 1000 μm, about 50 μm to about 750 μm,about 50 μm to about 500 μm, or about 100 μm to about 500 μm.

Further described herein are conjugates that are placed on the conjugatepad and immobilized to the conjugate pad until the sample is applied tothe support medium. The conjugates may comprise a nanoparticle, a goldnanoparticle, a latex nanoparticle, a quantum dot, a chemiluminescentnanoparticle, a carbon nanoparticle, a selenium nanoparticle, afluorescent nanoparticle, a liposome, or a dendrimer. The surface of theconjugate may be coated by a conjugate binding molecule that binds tothe detection moiety from the cleaved detector molecule.

The conjugate binding molecules described herein coat the surface of theconjugates and can bind to detection moiety. The conjugate bindingmolecule binds selectively to the detection moiety cleaved from thedetector nucleic acid. Some suitable conjugate binding moleculescomprise an antibody, a polypeptide, or a single stranded nucleic acid.In some cases, the conjugate binding molecule binds a dye and afluorophore. Some such conjugate binding molecules that bind to a dye ora fluorophore can quench their signal. In some cases, the conjugatebinding molecule is a monoclonal antibody. In some cases, an antibody,also referred to as an immunoglobulin, includes any isotype, variableregions, constant regions, Fc region, Fab fragments, F(ab′)2 fragments,and Fab′ fragments. Alternatively, the conjugate binding molecule is anon-antibody compound that specifically binds the detection moiety.Sometimes, the conjugate binding molecule is a polypeptide that can bindto the detection moiety. Sometimes, the conjugate binding molecule isavidin or a polypeptide that binds biotin. Sometimes, the conjugatebinding molecule is a detector moiety binding nucleic acid.

The diameter of the conjugate may be selected to provide a desiredsurface to volume ratio. In some instances, a high surface area tovolume ratio may allow for more conjugate binding molecules that areavailable to bind to the detection moiety per total volume of theconjugates. In some cases, the diameter of the conjugate may range fromabout 1 nm to about 1000 nm, about 1 nm to about 500 nm, about 1 nm toabout 100 nm, or about 1 nm to about 50 nm. In some cases, the diameterof the conjugate may be at least 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 30 nm, 35 nm, 40 nm,45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900nm, or 1000 nm. In some cases, the diameter of the conjugate may be nomore than 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm,15 nm, 20 nm, 25 nm, 30 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 200 nm, 300nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 1000 nm.

The ratio of conjugate binding molecules to the conjugates can betailored to achieve desired binding properties between the conjugatebinding molecules and the detection moiety. In some instances, the molarratio of conjugate binding molecules to the conjugates is at least 1:1,1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110,1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:250,1:300, 1:350, 1:400, 1:450, or 1:500. In some instances, the mass ratioof conjugate binding molecules to the conjugates is at least 1:1, 1:5,1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110,1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:250,1:300, 1:350, 1:400, 1:450, or 1:500. In some instances, the number ofconjugate binding molecules per conjugate is at least 1, 10, 50, 100,500, 1000, 5000, or 10000.

The conjugate binding molecules can be bound to the conjugates byvarious approached. Sometimes, the conjugate binding molecule can bebound to the conjugate by passive binding. Some such passive bindingcomprise adsorption, absorption, hydrophobic interaction, electrostaticinteraction, ionic binding, or surface interactions. In some cases, theconjugate binding molecule can be bound to the conjugate covalently.Sometimes, the covalent bonding of the conjugate binding molecule to theconjugate is facilitated by EDC/NHS chemistry or thiol chemistry.

Described herein are detection region on the support medium that providea region for presenting the assay results. The detection region can bemade from various materials that facilitate binding of theconjugate-bound detection moiety from cleaved detector molecule to thecapture molecule specific for the detection moiety. The detection padmay comprise the same material as other zones or a different materialthan the other zones. The detection region may comprise nitrocellulose,paper, cellulose, cellulose fiber filters, glass fiber filters, porousplastic membranes, aluminum oxide coated membranes, woven meshes,polyester filter, or polymer-based matrices. Often the detection regionmay comprise nitrocellulose. The material for the region pad region maybe hydrophilic, have low non-specific binding, or have consistent fluidflow properties across the region pad. The material for the conjugatepad may range from about 10 μm to about 1000 μm, about 10 μm to about750 μm, about 10 μm to about 500 μm, or about 10 μm to about 300 μm.

The detection region comprises at least one capture area with a highdensity of a capture molecule that can bind to the detection moiety fromcleaved detection molecule and at least one area with a high density ofa positive control capture molecule. The capture area with a highdensity of capture molecule or a positive control capture molecule maybe a line, a circle, an oval, a rectangle, a triangle, a plus sign, orany other shapes. In some instances, the detection region comprise morethan one capture area with high densities of more than one capturemolecules, where each capture area comprises one type of capturemolecule that specifically binds to one type of detection moiety fromcleaved detection molecule and are different from the capture moleculesin the other capture areas. The capture areas with different capturemolecules may be overlapping completely, overlapping partially, orspatially separate from each other. In some instances, the capture areasmay overlap and produce a combined detectable signal distinct from thedetectable signals generated by the individual capture areas. Usually,the positive control spot is spatially distinct from any of thedetection spot.

The capture molecule described herein bind to detection moiety andimmobilized in the detection spot in the detect region. Some suitablecapture molecules comprise an antibody, a polypeptide, or a singlestranded nucleic acid. In some cases, the capture molecule binds a dyeand a fluorophore. Some such capture molecules that bind to a dye or afluorophore can quench their signal. Sometimes, the capture molecule isan antibody that that binds to a dye or a fluorophore can quench theirsignal. In some cases, the capture molecule is a monoclonal antibody. Insome cases, an antibody, also referred to as an immunoglobulin, includesany isotype, variable regions, constant regions, Fc region, Fabfragments, F(ab′)2 fragments, and Fab′ fragments. Alternatively, thecapture molecule is a non-antibody compound that specifically binds thedetection moiety. Sometimes, the capture molecule is a polypeptide thatcan bind to the detection moiety. In some instances, the detectionmoiety from cleaved detection molecule has a conjugate bound to thedetection moiety, and the conjugate-detection moiety complex may bind tothe capture molecule specific to the detection moiety on the detectionregion. Sometimes, the capture molecule is a polypeptide that can bindto the detection moiety. Sometimes, the capture molecule is avidin or apolypeptide that binds biotin. Sometimes, the capture molecule is adetector moiety binding nucleic acid.

The detection region described herein comprises at least one area with ahigh density of a positive control capture molecule. The positivecontrol spot in the detection region provides a validation of the assayand a confirmation of completion of the assay. If the positive controlspot is not detectable by the visualization methods described herein,the assay is not valid and should be performed again with a new systemor kit. The positive control capture molecule binds at least one of theconjugate, the conjugate binding molecule, or detection moiety and isimmobilized in the positive control spot in the detect region. Somesuitable positive control capture molecules comprise an antibody, apolypeptide, or a single stranded nucleic acid. In some cases, thepositive control capture molecule binds to the conjugate bindingmolecule. Some such positive control capture molecules that bind to adye or a fluorophore can quench their signal. Sometimes, the positivecontrol capture molecule is an antibody that that binds to a dye or afluorophore can quench their signal. In some cases, the positive controlcapture molecule is a monoclonal antibody. In some cases, an antibodyincludes any isotype, variable regions, constant regions, Fc region, Fabfragments, F(ab′)2 fragments, and Fab′ fragments. Alternatively, thepositive control capture molecule is a non-antibody compound thatspecifically binds the detection moiety. Sometimes, the positive controlcapture molecule is a polypeptide that can bind to at least one of theconjugate, the conjugate binding molecule, or detection moiety. In someinstances, the conjugate unbound to the detection moiety binds to thepositive control capture molecule specific to at least one of theconjugate or the conjugate binding molecule.

The kit or system described herein may also comprise a positive controlsample to determine that the activity of at least one of programmablenuclease, a guide nucleic acid, or a single stranded detector nucleicacid. Often, the positive control sample comprises a target nucleic acidthat binds to the guide nucleic acid. The positive control sample iscontacted with the reagents in the same manner as the test sample andvisualized using the support medium. The visualization of the positivecontrol spot and the detection spot for the positive control sampleprovides a validation of the reagents and the assay.

The kit or system for detection of a target nucleic acid describedherein further comprises reagents for nucleic acid amplification oftarget nucleic acids in the sample. Isothermal nucleic acidamplification allows the use of the kit or system in remote regions orlow resource settings without specialized equipment for amplification.Often, the reagents for nucleic acid amplification comprise arecombinase, an oligonucleotide primer, a single-stranded DNA binding(SSB) protein, and a polymerase. Sometimes, nucleic acid amplificationof the sample improves at least one of sensitivity, specificity, oraccuracy of the assay in detecting the target nucleic acid. In somecases, the nucleic acid amplification is performed in a nucleic acidamplification region on the support medium. Alternatively, or incombination, the nucleic acid amplification is performed in a reagentchamber, and the resulting sample is applied to the support medium.Sometimes, the nucleic acid amplification is isothermal nucleic acidamplification. In some cases, the nucleic acid amplification istranscription mediated amplification (TMA). Nucleic acid amplificationis helicase dependent amplification (HDA) or circular helicase dependentamplification (cHDA) in other cases. In additional cases, nucleic acidamplification is strand displacement amplification (SDA). In some cases,nucleic acid amplification is by recombinase polymerase amplification(RPA). In some cases, nucleic acid amplification is by at least one ofloop mediated amplification (LAMP) or the exponential amplificationreaction (EXPAR). Nucleic acid amplification is, in some cases, byrolling circle amplification (RCA), ligase chain reaction (LCR), simplemethod amplifying RNA targets (SMART), single primer isothermalamplification (SPIA), multiple displacement amplification (MDA), nucleicacid sequence based amplification (NASBA), hinge-initiatedprimer-dependent amplification of nucleic acids (HIP), nicking enzymeamplification reaction (NEAR), or improved multiple displacementamplification (IMDA). Often, the nucleic acid amplification is performedfor no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes. Sometimes, thenucleic acid amplification reaction is performed at a temperature ofaround 20-45° C. In some cases, the nucleic acid amplification reactionis performed at a temperature no greater than 20° C., 25° C., 30° C.,35° C., 37° C., 40° C., 45° C. In some cases, the nucleic acidamplification reaction is performed at a temperature of at least 20° C.,25° C., 30° C., 35° C., 37° C., 40° C., or 45° C. Sometimes the nucleicacid amplification uses dTTP, dATP, dCTP, and dGTP. Often, the nucleicacid amplification uses dUTP, dATP, dCTP, and dGTP.

Described herein are collection pad region that provide a region tocollect the sample that flows down the support medium. Often thecollection pads are placed downstream of the detection region andcomprise an absorbent material. The collection pad can increase thetotal volume of sample that enters the support medium by collecting andremoving the sample from other regions of the support medium. Thisincreased volume can be used to wash unbound conjugates away from thedetection region to lower the background and enhance assay sensitivity.When the design of the support medium does not include a collection pad,the volume of sample analyzed in the support medium may be determined bythe bed volume of the support medium. The collection pad may provide areservoir for sample volume and may help to provide capillary force forthe flow of the sample down the support medium.

The collection pad may be prepared from various materials that arehighly absorbent and able to retain fluids. Often the collection padscomprise cellulose filters. In some instances, the collection padscomprise cellulose, cotton, woven meshes, polymer-based matrices. Thedimension of the collection pad, usually the length of the collectionpad, may be adjusted to change the overall volume absorbed by thesupport medium.

The support medium described herein may have a barrier around the edgeof the support medium. Often the barrier is a hydrophobic barrier thatfacilitates the maintenance of the sample within the support medium orflow of the sample within the support medium. Usually, the transportrate of the sample in the hydrophobic barrier is much lower than throughthe regions of the support medium. In some cases, the hydrophobicbarrier is prepared by contacting a hydrophobic material around the edgeof the support medium. Sometimes, the hydrophobic barrier comprises atleast one of wax, polydimethylsiloxane, rubber, or silicone.

Any of the regions on the support medium can be treated with chemicalsto improve the visualization of the detection spot and positive controlspot on the support medium. The regions can be treated to enhanceextraction of nucleic acid in the sample, to control the transport ofthe reacted reagents and sample or the conjugate to other regions of thesupport medium, or to enhance the binding of the cleaved detectionmoiety to the conjugate binding molecule on the surface of the conjugateor to the capture molecule in the detection region. The chemicals maycomprise detergents, surfactants, buffers, salts, viscosity enhancers,or polypeptides. In some instances, the chemical comprises bovine serumalbumin. In some cases, the chemicals or physical agents enhance flow ofthe sample with a more even flow across the width of the region. In somecases, the chemicals or physical agents provide a more even mixing ofthe sample across the width of the region. In some cases, the chemicalsor physical agents control flow rate to be faster or slower in order toimprove performance of the assay. Sometimes, the performance of theassay is measured by at least one of shorter assay time, longer timesduring cleavage activity, longer or shorter binding time with theconjugate, sensitivity, specificity, or accuracy.

Housing

A support medium as described herein can be housed in a number of waysthat are consistent with the methods disclosed herein. The housing forthe support medium are, for example, consistent with fluidic devicesdisclosed herein for detection of a target nucleic acid sequence withinthe sample, wherein the fluidic device may comprise multiple pumps,valves, reservoirs, and chambers for sample preparation, amplificationof a target nucleic acid sequence within the sample, mixing with aprogrammable nuclease, and detection of a detectable signal arising fromcleavage of detector nucleic acids by the programmable nuclease withinthe fluidic system itself. The housing, as described herein, arecompatible with the DETECTR assay methods disclosed herein. The housing,as described herein, are compatible with any of the programmablenucleases disclosed herein (e.g., a programmable nuclease with at least60% sequence identity to SEQ ID NO: 11) and use of said programmablenuclease in a method of detecting a target nucleic acid. The housing, asdescribed herein, are compatible with any of the compositions comprisinga programmable nuclease and a buffer, which has been developed toimprove the function of the programmable nuclease (e.g., a programmablenuclease and a buffer with low salt (about 110 mM or less) and a pH of 7to 8) and use of said compositions in a method of detecting a targetnucleic acid. The housing, as described herein, are compatible with anyof the methods disclosed herein including methods of assaying for atleast one base difference (e.g., assaying for a SNP or a base mutation)in a target nucleic acid sequence, methods of assaying for a targetnucleic acid that lacks a PAM by amplifying the target nucleic acidsequence to introduce a PAM, and compositions used in introducing a PAMvia amplification into the target nucleic acid sequence. For example,the fluidic device may comprise support mediums to channel the flow offluid from one chamber to another and wherein the entire fluidic deviceis encased within the housing described herein. Typically, the supportmedium described herein is encased in a housing to protect the supportmedium from contamination and from disassembly. The housing can be madeof more than one part and assembled to encase the support medium. Insome instances, a single housing can encase more than one supportmedium. The housing can be made from cardboard, plastics, polymers, ormaterials that provide mechanical protection for the support medium.Often, the material for the housing is inert or does not react with thesupport medium or the reagents placed on the support medium. The housingmay have an upper part which when in place exposes the sample pad toreceive the sample and has an opening or window above the detectionregion to allow the results of the lateral flow assay to be read. Thehousing may have guide pins on its inner surface that are placed aroundand on the support medium to help secure the compartments and thesupport medium in place within the housing. In some cases, the housingencases the entire support medium. Alternatively, the sample pad of thesupport medium is not encased and is left exposed to facilitate thereceiving of the sample while the rest of the support medium is encasedin the housing.

The housing and the support medium encased within the housing may besized to be small, portable, and hand held. The small size of thehousing and the support medium would facilitate the transport and use ofthe assay in remote regions or low resource settings. In some cases, thehousing has a length of no more than 30 cm, 25 cm, 20 cm, 15 cm, 10 cm,or 5 cm. In some cases, the housing has a length of at least 1 cm, 5 cm,10 cm, 15 cm, 20 cm, 25 cm, or 30 cm. In some cases, the housing has awidth of no more than 30 cm, 25 cm, 20 cm, 15 cm, 10 cm, 5 cm, 4 cm, 3cm, 2 cm, or 1 cm. In some cases, the housing has a width of at least 1cm, 2 cm, 3 cm, 4 cm, 5 cm, 10 cm, 15 cm, 20 cm, 25 cm, or 30 cm. Insome cases, the housing has a height of no more than 10 cm, 9 cm, 8 cm,7 cm, 6 cm, 5 cm, 4 cm, 3 cm, 2 cm, or 1 cm. In some cases, the housinghas a height of at least 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm,9 cm, or 10 cm. Typically, the housing is rectangular in shape.

In some instances, the housing provides additional information on theouter surface of the upper cover to facilitate the identification of thetest type, visualization of the detection region, and analysis of theresults. The upper outer housing may have identification label includingbut not limited to barcodes, QR codes, identification label, or othervisually identifiable labels. In some instances, the identificationlabel is imaged by a camera on a mobile device, and the image isanalyzed to identify the disease, cancer, or genetic disorder that isbeing tested for. The correct identification of the test is important toaccurately visualize and analyze the results. In some instances, theupper outer housing has fiduciary markers to orient the detection regionto distinguish the positive control spot from the detection spots. Insome instances, the upper outer housing has a color reference guide.When the detection region is imaged with the color reference guide, thedetection spots, located using the fiduciary marker, can be comparedwith the positive control spot and the color reference guide todetermine various image properties of the detection spot such as color,color intensity, and size of the spot. In some instances, the colorreference guide has red, green, blue, black, and white colors. In somecases, the image of the detection spot can be normalized to at least oneof the reference colors of the color reference guide, compared to atleast two of the reference colors of the color reference guide, andgenerate a value for the detection spot. Sometimes, the comparison to atleast two of the reference colors is comparison to a standard referencescale. In some instance, the image of the detection spot in someinstance undergoes transformation or filtering prior to analysis.Analysis of the image properties of the detection spot can provideinformation regarding presence or absence of the target nucleic acidtargeted by the assay and the disease, cancer, or genetic disorderassociated with the target nucleic acid. In some instances, the analysisprovides a qualitative result of presence or absence of the targetnucleic acid in the sample. In some instances, the analysis provides asemi-quantitative or quantitative result of the level of the targetnucleic acid present in the sample. Quantification may be performed byhaving a set of standards in spots/wells and comparing the test sampleto the range of standards. A more semi-quantitative approach may beperformed by calculating the color intensity of 2 spots/well compared toeach other and measuring if one spot/well is more intense than theother.

Manufacturing

The support medium may be assembled with a variety of materials andreagents. Reagents may be dispensed or coated on to the surface of thematerial for the support medium. The material for the support medium maybe laminated to a backing card, and the backing card may be singulatedor cut into individual test strips. The device may be manufactured bycompletely manual, batch-style processing; or a completely automated,in-line continuous process; or a hybrid of the two processingapproaches. The batch process may start with sheets or rolls of eachmaterial for the support medium. Individual zones of the support mediummay be processed independently for dispensing and drying, and the finalsupport medium may be assembled with the independently prepared zonesand cut. The batch processing scheme may have a lower cost of equipment,and a higher labor cost than more automated in-line processing, whichmay have higher equipment costs. In some instances, batch processing maybe preferred for low volume production due to the reduced capitalinvestment. In some instances, automated in-line processing may bepreferred for high volume production due to reduced production time.Both approaches may be scalable to production level.

In some instances, the support mediums are prepared using variousinstruments, including an XYZ-direction motion system with dispensers,impregnation tanks, drying ovens, a manual or semi-automated laminator,and cutting methods for reducing roll or sheet stock to appropriatelengths and widths for lamination. For dispensing the conjugate bindingmolecules for the conjugate zone and capture molecules for the detectionzones, an XYZ-direction motion system with dispensers may be used. Insome embodiments, the dispenser may dispense by a contact method or anon-contact method.

In automated or semi-automated preparation of the support medium, thesupport medium may be prepared from rolls of membranes for each regionthat are ordered into the final assembled order and unfurled from therolls. For example, the membranes can be ordered from sample pad regionto collection pad region from left to right with one membranecorresponding to a region on the support medium, all onto an adhesivecardstock. The dispenser places the reagents, conjugates, detectionmolecules, and other treatments for the membrane onto the membrane. Thedispensed fluids are dried onto the membranes by heat, in a low humiditychamber, or by freeze drying to stabilize the dispensed molecules. Themembranes are cut into strips and placed into the housing and packaged.

Kits

Disclosed herein are kits for use to detect a target nucleic acid asdisclosed herein using the methods as discuss above. In someembodiments, the kit comprises the programmable nuclease system,reagents, and the support medium. The reagents and programmable nucleasesystem can be provided in a reagent chamber or on the support medium.Alternatively, the reagent and programmable nuclease system can beplaced into the reagent chamber or the support medium by the individualusing the kit. Optionally, the kit further comprises a buffer and adropper. The reagent chamber can be a test well or container. Theopening of the reagent chamber can be large enough to accommodate thesupport medium. The buffer can be provided in a dropper bottle for easeof dispensing. The dropper can be disposable and transfer a fixedvolume. The dropper can be used to place a sample into the reagentchamber or on the support medium.

The kit or system for detection of a target nucleic acid describedherein further comprises reagents for nucleic acid amplification oftarget nucleic acids in the sample. Isothermal nucleic acidamplification allows the use of the kit or system in remote regions orlow resource settings without specialized equipment for amplification.Often, the reagents for nucleic acid amplification comprise arecombinase, a oligonucleotide primer, a single-stranded DNA binding(SSB) protein, and a polymerase. Sometimes, nucleic acid amplificationof the sample improves at least one of sensitivity, specificity, oraccuracy of the assay in detecting the target nucleic acid. In somecases, the nucleic acid amplification is performed in a nucleic acidamplification region on the support medium. Alternatively, or incombination, the nucleic acid amplification is performed in a reagentchamber, and the resulting sample is applied to the support medium.Sometimes, the nucleic acid amplification is isothermal nucleic acidamplification. In some cases, the nucleic acid amplification istranscription mediated amplification (TMA). Nucleic acid amplificationis helicase dependent amplification (HDA) or circular helicase dependentamplification (cHDA) in other cases. In additional cases, nucleic acidamplification is strand displacement amplification (SDA). In some cases,nucleic acid amplification is by recombinase polymerase amplification(RPA). In some cases, nucleic acid amplification is by at least one ofloop mediated amplification (LAMP) or the exponential amplificationreaction (EXPAR). Nucleic acid amplification is, in some cases, byrolling circle amplification (RCA), ligase chain reaction (LCR), simplemethod amplifying RNA targets (SMART), single primer isothermalamplification (SPIA), multiple displacement amplification (MDA), nucleicacid sequence based amplification (NASBA), hinge-initiatedprimer-dependent amplification of nucleic acids (HIP), nicking enzymeamplification reaction (NEAR), or improved multiple displacementamplification (IMDA). Often, the nucleic acid amplification is performedfor no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes, or any value from 1to 60 minutes. Sometimes, the nucleic acid amplification is performedfor from 1 to 60, from 5 to 55, from 10 to 50, from 15 to 45, from 20 to40, or from 25 to 35 minutes. Sometimes, the nucleic acid amplificationreaction is performed at a temperature of around 20-45° C. In somecases, the nucleic acid amplification reaction is performed at atemperature no greater than 20° C., 25° C., 30° C., 35° C., 37° C., 40°C., 45° C., or any value from 20° C. to 45° C. In some cases, thenucleic acid amplification reaction is performed at a temperature of atleast 20° C., 25° C., 30° C., 35° C., 37° C., 40° C., or 45° C., or anyvalue from 20° C. to 45° C. In some cases, the nucleic acidamplification reaction is performed at a temperature of from 20° C. to45° C., from 25° C. to 40° C., from 30° C. to 40° C., or from 35° C. to40° C.

In some embodiments, a kit for detecting a target nucleic acidcomprising a support medium; a guide nucleic acid targeting a targetsequence; a programmable nuclease capable of being activated whencomplexed with the guide nucleic acid and the target sequence; and adetector nucleic acid comprising a detection moiety, wherein thedetector nucleic acid is capable of being cleaved by the activatednuclease, thereby generating a first detectable signal. Often, the kitfurther comprises primers for amplifying a target nucleic acid ofinterest to produce a PAM target nucleic acid.

In some embodiments, a kit for detecting a target nucleic acidcomprising a PCR plate; a guide nucleic acid targeting a targetsequence; a programmable nuclease capable of being activated whencomplexed with the guide nucleic acid and the target sequence; and asingle stranded detector nucleic acid comprising a detection moiety,wherein the detector nucleic acid is capable of being cleaved by theactivated nuclease, thereby generating a first detectable signal. Thewells of the PCR plate can be pre-aliquoted with the guide nucleic acidtargeting a target sequence, a programmable nuclease capable of beingactivated when complexed with the guide nucleic acid and the targetsequence, and at least one population of a single stranded detectornucleic acid comprising a detection moiety. A user can thus add thebiological sample of interest to a well of the pre-aliquoted PCR plateand measure for the detectable signal with a fluorescent light reader ora visible light reader.

In some instances, such kits may include a package, carrier, orcontainer that is compartmentalized to receive one or more containerssuch as vials, tubes, and the like, each of the container(s) comprisingone of the separate elements to be used in a method described herein.Suitable containers include, for example, test wells, bottles, vials,and test tubes. In one embodiment, the containers are formed from avariety of materials such as glass, plastic, or polymers.

The kit or systems described herein contain packaging materials.Examples of packaging materials include, but are not limited to,pouches, blister packs, bottles, tubes, bags, containers, bottles, andany packaging material suitable for intended mode of use.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included. In one embodiment, a labelis on or associated with the container. In some instances, a label is ona container when letters, numbers or other characters forming the labelare attached, molded or etched into the container itself; a label isassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. In oneembodiment, a label is used to indicate that the contents are to be usedfor a specific therapeutic application. The label also indicatesdirections for use of the contents, such as in the methods describedherein.

After packaging the formed product and wrapping or boxing to maintain asterile barrier, the product may be terminally sterilized by heatsterilization, gas sterilization, gamma irradiation, or by electron beamsterilization. Alternatively, the product may be prepared and packagedby aseptic processing.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. As used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. Any referenceto “or” herein is intended to encompass “and/or” unless otherwisestated.

As used herein, the term “comprising” and its grammatical equivalentsspecifies the presence of stated features, integers, steps, operations,elements, and/or components, but do not preclude the presence oraddition of one or more other features, integers, steps, operations,elements, components, and/or groups thereof. As used herein, the term“and/or” includes any and all combinations of one or more of theassociated listed items.

Unless specifically stated or obvious from context, as used herein, theterm “about” in reference to a number or range of numbers is understoodto mean the stated number and numbers+/−10% thereof, or 10% below thelower listed limit and 10% above the higher listed limit for the valueslisted for a range.

As used herein the terms “individual,” “subject,” and “patient” are usedinterchangeably and include any member of the animal kingdom, includinghumans.

As used herein the term “antibody” refers to, but not limited to, amonoclonal antibody, a synthetic antibody, a polyclonal antibody, amultispecific antibody (including a bi-specific antibody), a humanantibody, a humanized antibody, a chimeric antibody, a single-chain Fvs(scFv) (including bi-specific scFvs), a single chain antibody, a Fabfragment, a F(ab′) fragment, a disulfide-linked Fvs (sdFv), or anepitope-binding fragment thereof. In some cases, the antibody is animmunoglobulin molecule or an immunologically active portion of animmunoglobulin molecule. In some instances, an antibody is animal inorigin including birds and mammals. Alternately, an antibody is human ora humanized monoclonal antibody.

At FIG. 1, one sees an improved SNP detection enzyme and method. At leftis shown ALDH2 E540K G-SNP, while at right one sees E540K A-SNP. TheALDH2 G-SNP was detected with a G-SNP gRNA (SEQ ID NO: 425), and theALDH2 A-SNP was detected with an A-SNP gRNA (SEQ ID NO: 426). LbCas12a(SEQ ID NO: 1) is shown at top, while a representative improved enzyme,a Cas12 variant corresponding to (SEQ ID NO: 11), is shown at bottom.One sees that the improved enzyme exhibits at least a 50% improvement inreaching reporter saturation signal, and exhibits no more than 33% offtarget reporter signal. At right, one sees that the improvement inreaching reporter saturation signal is at least 2×, and the off targetreporter signal is no greater than 10% of the target signal.

Various compositions and implementations of the methods herein achievean improvement in reaching reporter signal saturation of at least 50%,60%, 70%, 80%, 90%, 2×, 2.5×, 3×, 3.5×, 4×, or more than 4×, or anyimprovement spanned by or greater than the range of improvements listedherein. Similarly, off target signal strength is observed to be nogreater than 33%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or lessthan 1%, or any value spanned by or less than the range of improvementslisted herein.

At FIG. 2, one sees the first of a series of experiments to assessbuffer contents for detection using a Cas12 variant (SEQ ID NO: 11). Itis observed that BIS-TRIS at pH 7.0, Imidazole at pH of 7.0, 7.5 or 7.8,MOPS at 7.0, HEPES at pH 7.0 or 7.5, and DIPSO at pH 7.0 exhibit topperformance.

Accordingly, disclosed herein are buffers comprising at least one of thecomponents listed above, at a pH such as a neutral pH, for example a pHranging from 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5, or any number fallingwithin or adjacent to the range defined thereby.

At FIG. 3, one sees improvements conveyed by inclusion of acetate atconcentrations of about 0, 10, 20, 37, 75, 150, 300 and 600 mM, fromleft to right on detection using a Cas12 variant (SEQ ID NO: 11). Theleft bar at each concentration is Cl and right bar is acetate.

Accordingly, one sees benefits conveyed by modulation of saltconcentration, for example by addition of acetate, as well as limitingsalt concentration to no greater than 10, 20, 37, 75, 150, 300 and 600mM. Particular improvements are seen at less than 75 nM, at no greaterthan 40 nM, and at about 10-20 nM. Disclosed herein are compositionshaving a reduced salt concentration, such as a salt concentration in nMof no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 669,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140,150, 160, 170, 18, 190, 200, 220, 230, 240, 250, 260, 270, 280, 290, or300.

At FIG. 4, one sees an improvement in SNP specificity upon inclusion ofheparin in a reaction buffer when detected with a Cas12 variant (SEQ IDNO: 11). Inclusion of heparin increases both SNP-specific detection andgeneral enzyme performance.

At FIG. 5, one sees optimization for a number of buffer additives, suchas heparin, DTT, NP-40, and BSA (from left to right) over a series of 8iterative dilutions when detected with a Cas12 variant (SEQ ID NO: 11)and a gRNA of SEQ ID NO: 423.

Accordingly, buffers comprising at least one additive selected from thegroup consisting of heparin, DTT, NP-40, and BSA, and optionallyincluding in addition or in the alternative triton X, are disclosedherein.

At FIG. 6, one sees base sensitivity for each SNP allele, A, C, G, or T,at a SNP position, for LbCas12a (SEQ ID NO: 1), top, and arepresentative improved enzyme, a Cas12 variant corresponding to (SEQ IDNO: 11), below. One sees substantial improvement over LbCas12a. Targetsequences corresponding to SEQ ID NO: 431-SEQ ID NO: 438, provided inTABLE 7 were detected. The A SNP allele was detected using a gRNA of SEQID NO: 427. The C SNP allele was detected using a gRNA of SEQ ID NO:428. The G SNP allele was detected using a gRNA of SEQ ID NO: 429. The TSNP allele was detected using a gRNA of SEQ ID NO: 430.

TABLE 7 Target and Non-Target Strands for SNP Allele SensitivitySEQ ID NO: Name Sequence SEQ ID NO: T-SNP TargetTGGTATTCTTTCTCTTCCGCACCCAGCAGTTTGGCCaG 431 Strand CCCAAAATCTGTGATCTSEQ ID NO: T-SNP Non-Target AGATCACAGATTTTGGGCtGGCCAAACTGCTGGGTGC 432Strand GGAAGAGAAAGAATACCA SEQ ID NO: G-SNP TargetTGGTATTCTTTCTCTTCCGCACCCAGCAGTTTGGCCcG 433 Strand CCCAAAATCTGTGATCTSEQ ID NO: G-SNP Non-Target AGATCACAGATTTTGGGCgGGCCAAACTGCTGGGTGC 434Strand GGAAGAGAAAGAATACCA SEQ ID NO: C-SNP TargetTGGTATTCTTTCTCTTCCGCACCCAGCAGTTTGGCCgG 435 Strand CCCAAAATCTGTGATCTSEQ ID NO: C-SNP Non-Target AGATCACAGATTTTGGGCcGGCCAAACTGCTGGGTGC 436Strand GGAAGAGAAAGAATACCA SEQ ID NO: A-SNP TargetTGGTATTCTTTCTCTTCCGCACCCAGCAGTTTGGCCtG 437 Strand CCCAAAATCTGTGATCTSEQ ID NO: A-SNP Non-Target AGATCACAGATTTTGGGCaGGCCAAACTGCTGGGTGC 438Strand GGAAGAGAAAGAATACCA

At FIG. 7, one sees template optimization for an improved enzyme, aCas12 variant corresponding to SEQ ID NO: 11, as disclosed herein.Templates comprising a C SNP allele (SEQ ID NO: 440) or a T SNP allele(SEQ ID NO: 441) were detected using gRNAs directed to the C SNP (SEQ IDNO: 423) or the T SNP allele (SEQ ID NO: 439). Primers corresponding toSEQ ID NO: 396 and SEQ ID NO: 397 were used to amplify the targetsequence and insert a PAM sequence.

At FIG. 8, one sees base sensitivity of an improved enzyme, a Cas12variant corresponding to SEQ ID NO: 11, for each SNP allele, A, C, G, orT, for an EGFR SNP as disclosed herein. EGFR target sequencescorresponding to SEQ ID NO: 444-SEQ ID NO: 447, provided in TABLE 8,were detected. Primers corresponding to SEQ ID NO: 442 and SEQ ID NO:443 were used to amplify the target sequences. The A SNP allele wasdetected using a gRNA of SEQ ID NO: 427. The C SNP allele was detectedusing a gRNA of SEQ ID NO: 428. The G SNP allele was detected using agRNA of SEQ ID NO: 429. The T SNP allele was detected using a gRNA ofSEQ ID NO: 430.

TABLE 8 EGFR Target Sequences SEQ ID NO: Name Sequence SEQ ID NO:EGFR WT TGGTCCCCGCCACCCCCCACCCCCACTTTGCAGATAAACCACATG 444 (G-SNP)CAGGAAGGTCAGCCTGGCAAGTCCAGTAAGTTCAAGCCCAGGTCTCAACTGGGCAGCAGAGCTCCTGCTCTTCTTTGTCCTCATATACGAGCACCTCTGGACTTAAAACTTGAGGAACTGGATGGAGAAAAGTTAATGGTCAGCAGCGGGTTACATCTTCTTTCATGCGCCTTTCCATTCTTTGGATCAGTAGTCACTAACGTTCGCCAGCCATAAGTCCTCGACGTGGAGAGGCTCAGAGCCTGGCATGAACATGACCCTGAATTCGGATGCAGAGCTTCTTCCCATGATGATCTGTCCCTCACAGCAGGGTCTTCTCTGTTTCAGGGCATGAACTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAGTAAGGAGGTGGCTTTAGGTCAGCCAGCATTTTCCTGACACCAGGGACCAGGCTGCCTTCCCACTAGCTGTATTGTTTAACACATGCAGGGGAGGATGCTCTCCAGACATTCTGGGTGAGCTCGCAGCAGCTGCTGCTGGCAGCTGGGTCCAGCCAGGGTCTCCTGGTAGTGTGAGCCAGAGCTGCTTTGGGAACAGTACTTGCTGGGACAGTGAATGAGGATGTTATCCCCAGGTGATCATTAGCAAATGTTAGGTTTCAGTCTCTCCCTGCAGGATATATAAGTCCCCTTCAATAGCGCAATTGGGAAAGGTCACAGCTGCCTTGGTGGTCCACTGCTGTCAAGGACACCTAAGGAACAGGAAAGGCCCCATGCGGACCCGAGCTCCCAGGGCTGTCTGTGGCTCGTGGCTGGGACAGGCAGCAATGGAGTCCTTCTCTCCCTTCACTGGCTCGG TTTCT SEQ ID NO: EGFR A-TGGTCCCCGCCACCCCCCACCCCCACTTTGCAGATAAACCACATG 445 SNPCAGGAAGGTCAGCCTGGCAAGTCCAGTAAGTTCAAGCCCAGGTCTCAACTGGGCAGCAGAGCTCCTGCTCTTCTTTGTCCTCATATACGAGCACCTCTGGACTTAAAACTTGAGGAACTGGATGGAGAAAAGTTAATGGTCAGCAGCGGGTTACATCTTCTTTCATGCGCCTTTCCATTCTTTGGATCAGTAGTCACTAACGTTCGCCAGCCATAAGTCCTCGACGTGGAGAGGCTCAGAGCCTGGCATGAACATGACCCTGAATTCGGATGCAGAGCTTCTTCCCATGATGATCTGTCCCTCACAGCAGGGTCTTCTCTGTTTCAGGGCATGAACTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCAGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAGTAAGGAGGTGGCTTTAGGTCAGCCAGCATTTTCCTGACACCAGGGACCAGGCTGCCTTCCCACTAGCTGTATTGTTTAACACATGCAGGGGAGGATGCTCTCCAGACATTCTGGGTGAGCTCGCAGCAGCTGCTGCTGGCAGCTGGGTCCAGCCAGGGTCTCCTGGTAGTGTGAGCCAGAGCTGCTTTGGGAACAGTACTTGCTGGGACAGTGAATGAGGATGTTATCCCCAGGTGATCATTAGCAAATGTTAGGTTTCAGTCTCTCCCTGCAGGATATATAAGTCCCCTTCAATAGCGCAATTGGGAAAGGTCACAGCTGCCTTGGTGGTCCACTGCTGTCAAGGACACCTAAGGAACAGGAAAGGCCCCATGCGGACCCGAGCTCCCAGGGCTGTCTGTGGCTCGTGGCTGGGACAGGCAGCAATGGAGTCCTTCTCTCCCTTCACTGGCTCGG TTTCT SEQ ID NO: EGFR T-TGGTCCCCGCCACCCCCCACCCCCACTTTGCAGATAAACCACATG 446 SNPCAGGAAGGTCAGCCTGGCAAGTCCAGTAAGTTCAAGCCCAGGTCTCAACTGGGCAGCAGAGCTCCTGCTCTTCTTTGTCCTCATATACGAGCACCTCTGGACTTAAAACTTGAGGAACTGGATGGAGAAAAGTTAATGGTCAGCAGCGGGTTACATCTTCTTTCATGCGCCTTTCCATTCTTTGGATCAGTAGTCACTAACGTTCGCCAGCCATAAGTCCTCGACGTGGAGAGGCTCAGAGCCTGGCATGAACATGACCCTGAATTCGGATGCAGAGCTTCTTCCCATGATGATCTGTCCCTCACAGCAGGGTCTTCTCTGTTTCAGGGCATGAACTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAGTAAGGAGGTGGCTTTAGGTCAGCCAGCATTTTCCTGACACCAGGGACCAGGCTGCCTTCCCACTAGCTGTATTGTTTAACACATGCAGGGGAGGATGCTCTCCAGACATTCTGGGTGAGCTCGCAGCAGCTGCTGCTGGCAGCTGGGTCCAGCCAGGGTCTCCTGGTAGTGTGAGCCAGAGCTGCTTTGGGAACAGTACTTGCTGGGACAGTGAATGAGGATGTTATCCCCAGGTGATCATTAGCAAATGTTAGGTTTCAGTCTCTCCCTGCAGGATATATAAGTCCCCTTCAATAGCGCAATTGGGAAAGGTCACAGCTGCCTTGGTGGTCCACTGCTGTCAAGGACACCTAAGGAACAGGAAAGGCCCCATGCGGACCCGAGCTCCCAGGGCTGTCTGTGGCTCGTGGCTGGGACAGGCAGCAATGGAGTCCTTCTCTCCCTTCACTGGCTCGG TTTCT SEQ ID NO: EGFR C-TGGTCCCCGCCACCCCCCACCCCCACTTTGCAGATAAACCACATG 447 SNPCAGGAAGGTCAGCCTGGCAAGTCCAGTAAGTTCAAGCCCAGGTCTCAACTGGGCAGCAGAGCTCCTGCTCTTCTTTGTCCTCATATACGAGCACCTCTGGACTTAAAACTTGAGGAACTGGATGGAGAAAAGTTAATGGTCAGCAGCGGGTTACATCTTCTTTCATGCGCCTTTCCATTCTTTGGATCAGTAGTCACTAACGTTCGCCAGCCATAAGTCCTCGACGTGGAGAGGCTCAGAGCCTGGCATGAACATGACCCTGAATTCGGATGCAGAGCTTCTTCCCATGATGATCTGTCCCTCACAGCAGGGTCTTCTCTGTTTCAGGGCATGAACTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCCGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAGTAAGGAGGTGGCTTTAGGTCAGCCAGCATTTTCCTGACACCAGGGACCAGGCTGCCTTCCCACTAGCTGTATTGTTTAACACATGCAGGGGAGGATGCTCTCCAGACATTCTGGGTGAGCTCGCAGCAGCTGCTGCTGGCAGCTGGGTCCAGCCAGGGTCTCCTGGTAGTGTGAGCCAGAGCTGCTTTGGGAACAGTACTTGCTGGGACAGTGAATGAGGATGTTATCCCCAGGTGATCATTAGCAAATGTTAGGTTTCAGTCTCTCCCTGCAGGATATATAAGTCCCCTTCAATAGCGCAATTGGGAAAGGTCACAGCTGCCTTGGTGGTCCACTGCTGTCAAGGACACCTAAGGAACAGGAAAGGCCCCATGCGGACCCGAGCTCCCAGGGCTGTCTGTGGCTCGTGGCTGGGACAGGCAGCAATGGAGTCCTTCTCTCCCTTCACTGGCTCGG TTTCT

At FIG. 9, one sees an assessment of buffer additives and their effecton detection using a Cas12 variant (SEQ ID NO: 11). Highest performingadditives include 4 M DMSO, 1M Pyridine, 500 mM polypropylene glycol400, 100 mM dithiothreitol, 2M Erythritol, 500 mM, polyethylene glycol,100 w/v polyvinyl alcohol type II, and 5% w/v polyvinylpyrrolidone k15.

Accordingly, disclosed here are buffers supplemented or comprising atleast one component selected from the list comprising DMSO, Pyridine,polypropylene glycol 400, dithiothreitol, Erythritol, polyethyleneglycol, polyvinyl alcohol type II, and 5 polyvinylpyrrolidone k15.Concentrations are contemplated to be about or exactly the valuespresented above, such as 0, 1, 2, 3, 4, 5, 6, 7, or 8 M, or anyinterceding value in the range defined thereby, or 0, 100, 200, 300,400, 500, 600, 700, or 800 mM or any interceding value in the rangedefined thereby, or 0, 1, 2, 3, 4, 5, 6, 7, or 80% w/v, or anyinterceding value in the range defined thereby.

At FIG. 10, one sees trans cleavage activity of various Cas12 orthologsor other improved enzymes corresponding to SEQ ID NO: 586, SEQ ID NO:581, SEQ ID NO: 576, SEQ ID NO: 587, SEQ ID NO: 578, SEQ ID NO: 572, SEQID NO: 575, SEQ ID NO: 11, SEQ ID NO: 573, SEQ ID NO: 589, and SEQ IDNO: 583, and of LbCas12a (SEQ ID NO: 1) on targets containing variousPAMs, double and single mismatched substrates. Shading indicates thebackground subtracted fluorescence signal. NTS, single-strandednon-target substrate, TS, single-stranded target substrate; OFF, anoff-target substrate; MM, location of a base mismatch.

Accordingly, disclosed herein are improved enzymes and associated kitsand methods relating to enzymes having tolerance for or sensitivity to aparticular PAM sequence or to a particular location of a mismatch, orboth a PAM sequence and a particular location for a mismatch.

At FIG. 11, one sees trans cleavage activity of various Cas12 orthologsor other improved enzymes corresponding to SEQ ID NO: 2, SEQ ID NO: 1,SEQ ID NO: 590-SEQ ID NO: 598, SEQ ID NO: 580, and SEQ ID NO: 599-SEQ IDNO: 602 on targets containing various PAMs, double and single mismatchedsubstrates. Shading indicates the background subtracted fluorescencesignal. JSC142, AsCas12a; JSC143, LbCas12a; pLBH835, MAD7.

At FIG. 12, one sees trans cleavage activity of various Cas12 orthologsor other improved enzymes corresponding to SEQ ID NO: 571-SEQ ID NO:577, SEQ ID NO: 11, SEQ ID NO: 578-SEQ ID NO: 589, SEQ ID NO: 1, and SEQID NO: 3 on targets containing various PAMs, double and singlemismatched substrates. Shading indicates the background subtractedfluorescence normalized to the maximum value for each.

At FIG. 13A, FIG. 13B, and FIG. 13C, one sees trans cleavage activity ofvarious Cas12 orthologs corresponding SEQ ID NO: 571-SEQ ID NO: 577, SEQID NO: 11, SEQ ID NO: 578-SEQ ID NO: 589, SEQ ID NO: 1, and SEQ ID NO: 3on PCR targets containing a TTTA (SEQ ID NO: 384) PAM using variousguide RNA repeat sequences. Shading indicates the background subtractedfluorescence normalized to the maximum value for each. Each plotrepresents an independent replicate. Activity was detected in thepresence of different Cas12 variants and different pre-crRNAscorresponding to different Cas12 variants. Sequences of the pre-crRNAsare provided in TABLE 30.

At FIG. 14, one sees activity of various Cas12 orthologs and otherimproved enzymes corresponding to SEQ ID NO: 571-SEQ ID NO: 577, SEQ IDNO: 11, SEQ ID NO: 578-SEQ ID NO: 589, SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 590-SEQ ID NO: 598, SEQ ID NO: 580, SEQ ID NO: 599-SEQ ID NO:602, and SEQ ID NO: 2 on a target PCR product. The negative control(“(−) control”) is PCR product with no Cas12 added. The positive controlis cleavage with a BamHI restriction enzyme (“BamHI”). Numbers aboveeach lane correspond to the time in minutes before the reaction wasquenched with 10 mM EDTA. Lanes marked with “−” under each Cas12ortholog correspond to negative control conditions with protein but nocrRNA.

At FIG. 15, one sees limit of detection (LOD) assay results indicatingtrans cleavage activity of various Cas12 orthologs or other improvedenzymes corresponding to SEQ ID NO: 572, SEQ ID NO: 576, SEQ ID NO: 11,SEQ ID NO: 582, SEQ ID NO: 583, SEQ ID NO: 587, SEQ ID NO: 1, SEQ ID NO:591, SEQ ID NO: 595, SEQ ID NO: 597, SEQ ID NO: 600, SEQ ID NO: 601, andSEQ ID NO: 2 in the presence of various activator concentrations (shownon the left). Shading indicates the background subtracted fluorescencevalue of after 90 min.

Accordingly, one sees improved enzymes, kits and methods exhibitingsensitivity of as low as 1 nM, 100 pM, 10 pM, 1 pM, 100 fM, 10 fM, or 1fM, or any number spanned by the range define thereby.

At FIG. 16A and FIG. 16B, one sees trans cleavage activity of variousCas12 orthologs corresponding to SEQ ID NO: 590-SEQ ID NO: 598, SEQ IDNO: 580, SEQ ID NO: 599-SEQ ID NO: 602, and SEQ ID NO: 2 in the presenceof various salt concentrations. The shading represents the backgroundsubtracted fluorescence normalized to the maximum value for thatprotein. NaCl concentrations are given for the amount of salt added tothe reaction for the added water (eg 0 mM=40 mM final saltconcentration).

At FIG. 17A and FIG. 17B, one sees trans cleavage activity of variousCas12 orthologs corresponding to SEQ ID NO: 590-SEQ ID NO: 598, SEQ IDNO: 580, SEQ ID NO: 599-SEQ ID NO: 602, and SEQ ID NO: 2 in the presenceof various salt concentrations. The color represents the raw backgroundsubtracted fluorescence (no normalization). NaCl concentrations aregiven for the amount of salt added to the reaction for the added water(e.g., 0 mM=40 mM final salt concentration).

Accordingly, disclosed herein are compositions supporting Cas12 or otherimproved enzyme activity and having reduced salt concentrations, such aslimiting salt concentration to no greater than 10, 20, 37, 75, 150, 300and 600 mM. Particular improvements are seen at less than 75 nM, at nogreater than 40 nM, and at about 10-20 nM. Disclosed herein arecompositions having a reduced salt concentration, such as a saltconcentration in nM of no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 669, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,110, 120, 130, 140, 150, 160, 170, 18, 190, 200, 220, 230, 240, 250,260, 270, 280, 290, or 300.

Numbered Embodiments

The following embodiments recite non-limiting permutations ofcombinations of features disclosed herein. Other permutations ofcombinations of features are also contemplated. In particular, each ofthese numbered embodiments is contemplated as depending from or relatingto every previous or subsequent numbered embodiment, independent oftheir order as listed. 1. A programmable nuclease that elicits maximalreporter activity no more than 60 minutes following contacting to atarget template at a target template concentration of 100 nM. 2. Theprogrammable nuclease of embodiment 1, wherein the programmable nucleasecomprises a Cas12 protein, a Cas13 protein, or a Cas14 protein. 3. TheCas12 protein of any one of embodiments 1-2, wherein said proteinelicits maximal reporter activity following contacting to a targettemplate at least 50% faster than LbCas12 at a given target templateconcentration. 4. The Cas12 protein of any one of embodiments 1-3,wherein said protein elicits maximal reporter activity followingcontacting to a target template at least 2× faster than LbCas12 at agiven target template concentration. 5. The Cas12 protein of any one ofembodiments 1-4, wherein said protein elicits maximal reporter activityfollowing contacting to a target template at least 4× faster thanLbCas12 at a given target template concentration. 6. The Cas12 proteinof any one of embodiments 1-5, wherein said protein elicits no greaterthan 33% of maximal reporter activity following contacting to a templatediffering from a target template by a single base at a templateconcentration of 100 nM. 7. The Cas12 protein of any one of embodiments1-6, wherein the protein elicits maximal reporter activity in acomposition comprising at least one component selected from the listconsisting of acetate, heparin, dithiothreitol (DTT), triton-X, TCEP,BSA, NP-40, imidazole, MOPS, HEPES and DIPSO. 8. The Cas12 protein ofany one of embodiments 1-7, wherein the template is unamplified. 9. TheCas12 protein of any one of embodiments 1-8, wherein the template isamplified prior to contacting. 10. The Cas12 protein of any one ofembodiments 1-9, wherein the contacting is performed in an activitybuffer, wherein the activity buffer comprises 125 mM NaCl, 5 mM MgCl2,20 mM Tris pH 7.5, and 1% glycerol. 11. The Cas12 protein of any one ofembodiments 1-10, wherein the contacting is performed at about 25° C.12. The Cas12 protein of any one of embodiments 1-11, wherein thecontacting is performed at about 37° C. 13. A programmable nucleasereaction buffer comprising at least one component selected from the listconsisting of acetate, heparin, dithiothreitol (DTT), triton-X, TCEP,BSA, NP-40, imidazole, MOPS, HEPES and DIPSO. 14. The programmablenuclease of any one of embodiments 1-13, wherein the programmablenuclease comprises a Cas12 protein, a Cas13 protein, or a Cas14 protein.15. The reaction buffer of any one of embodiments 13-14, wherein theprogrammable nuclease in said reaction buffer elicits no greater than33% of maximal reporter activity following contacting to a templatediffering from a target template by a single base. 16. The reactionbuffer of any one of embodiments 13-15, wherein the reaction buffercomprises no greater than 150 mM NaCl. 17. The reaction buffer of anyone of embodiments 13-16, wherein the reaction buffer comprises nogreater than 100 mM NaCl. 18. The reaction buffer of any one ofembodiments 13-17, wherein the reaction buffer comprises no greater than50 mM NaCl. 19. The reaction buffer of any one of embodiments 13-18,wherein the reaction buffer comprises no greater than 25 mM NaCl. 20.The reaction buffer of any one of embodiments 13-19, wherein thereaction buffer comprises from 0 μg/mL heparin to 100 μg/mL heparin. 21.The reaction buffer of any one of embodiments 13-20, wherein thereaction buffer comprises 0 μg/mL heparin. 22. The reaction buffer ofany one of embodiments 13-21, wherein the reaction buffer comprises 50μg/mL heparin. 23. The reaction buffer of any one of embodiments 13-22,wherein the reaction buffer comprises from 0 mM DTT to 5 mM DTT. 24. Thereaction buffer of any one of embodiments 13-23, wherein the reactionbuffer comprises 1 mM DTT. 25. The reaction buffer of any one ofembodiments 13-24, wherein the reaction buffer comprises from 0 mM to 50mM Imidazole. 26. The reaction buffer of any one of embodiments 13-25,wherein the reaction buffer comprises 20 mM Imidazole. 27. Aprogrammable nuclease reaction buffer comprising at least one componentselected from the list consisting of DMSO, polyvinyl alcohol,polyvinylpyrrolidone, and polypropylene glycol. 28. The programmablenuclease of any one of embodiments 1-27, wherein the programmablenuclease comprises a Cas12 protein, a Cas13 protein, or a Cas14 protein.29. The reaction buffer of any one of embodiments 13-28, wherein theprogrammable nuclease in said reaction buffer elicits no greater than33% of maximal reporter activity following contacting to a no-templatecontrol. 30. The reaction buffer of any one of embodiments 13-29,wherein the reaction buffer comprises no greater than 150 mM NaCl. 31.The reaction buffer of any one of embodiments 13-30, wherein thereaction buffer comprises no greater than 100 mM NaCl. 32. The reactionbuffer of any one of embodiments 13-31, wherein the reaction buffercomprises no greater than 50 mM NaCl. 33. The reaction buffer of any oneof embodiments 13-32, wherein the reaction buffer comprises no greaterthan 25 mM NaCl. 34. A programmable nuclease that elicits reporteractivity no more than 60 minutes following contacting to a targettemplate at a target template concentration of 1 nM in an activitybuffer, wherein the activity buffer comprises 125 mM NaCl, 5 mM MgCl2,20 mM Tris pH 7.5, and 1% glycerol. 35. The programmable nuclease of anyone of embodiments 1-34, wherein the programmable nuclease comprises aCas12 protein, a Cas13 protein, or a Cas14 protein. 36. The Cas12protein of any one of embodiments 1-35, wherein the Cas12 proteinelicits reporter activity no more than 60 minutes following contactingto a target template at a target template concentration of 1 pM. 37. TheCas12 protein of any one of embodiments 1-36, wherein the Cas12 proteinelicits reporter activity no more than 60 minutes following contactingto a target template at a target template concentration of 1 fM. 38. Aprogrammable nuclease that exhibits at least 90% target cleavage in nomore than 60 minutes. 39. The programmable nuclease of any one ofembodiments 1-38, wherein the programmable nuclease comprises a Cas12protein, a Cas13 protein, or a Cas14 protein. 40. The Cas12 protein ofany one of embodiments 1-39, wherein the Cas12 protein exhibits at least90% target cleavage in no more than 15 minutes. 41. The Cas12 protein ofany one of embodiments 1-40, wherein an activity buffer (5×: 600 mMNaCl, 25 mM MgCl2, 100 mM Tris pH 7.5, 5% glycerol) exhibits said targetcleavage. 42. The Cas12 protein of any one of embodiments 1-41, whereinsaid cleavage is effected at a Cas12 concentration of from 50 nM to 200nM. 43. The Cas12 protein of any one of embodiments 1-42, wherein saidtarget cleavage is effected at a Cas12 concentration of 100 nM. 44. TheCas12 protein of any one of embodiments 1-42, wherein said cleavage iseffected at a target concentration of from 5 nm to 25 nM. 45. The Cas12protein of any one of embodiments 1-44, wherein said target cleavage iseffected at a target concentration of 15 nM. 46. The Cas12 protein ofany one of embodiments 1-45, wherein said target cleavage is effected ata guide RNA concentration of from 50 nM to 200 nM. 47. The Cas12 proteinof any one of embodiments 1-46, wherein said target cleavage is effectedat a guide RNA concentration of 125 nM. 48. The Cas12 protein of any oneof embodiments 1-47, wherein said target cleavage is effected at atemperature of from about 20° C. to about 40° C. 49. The Cas12 proteinof any one of embodiments 1-48, wherein said target cleavage is effectedat a temperature of about 25° C. 50. The Cas12 protein of any one ofembodiments 1-49, wherein said target cleavage is effected at atemperature of about 37° C. 51. A programmable nuclease that exhibits nomore than 10% target cleavage in 60 minutes. 52. The programmablenuclease of any one of embodiments 1-51, wherein the programmablenuclease comprises a Cas12 protein, a Cas13 protein, or a Cas14 protein.53. The Cas12 protein of any one of embodiments 1-52, wherein theprogrammable nuclease exhibits said target cleavage in an activitybuffer comprising 125 mM NaCl, 5 mM MgCl2, 20 mM Tris pH 7.5, and 1%glycerol. 54. The Cas12 protein of any one of embodiments 1-53, whereinsaid target cleavage is effected at a Cas12 concentration of 100 nM. 55.The Cas12 protein of any one of embodiments 1-54, wherein said targetcleavage is effected at a target concentration of 15 nM. 56. The Cas12protein of any one of embodiments 1-55, wherein said target cleavage iseffected at a guide RNA concentration of 125 nM. 57. The Cas12 proteinof any one of embodiments 1-56, wherein said target cleavage is effectedat a temperature of about 25° C. 58. The Cas12 protein of any one ofembodiments 1-57, wherein said target cleavage is effected at atemperature of about 37° C. 59. A composition comprising a firstprogrammable nuclease population and a second programmable nucleasepopulation, wherein the first programmable nuclease population and thesecond programmable nuclease population do not recognize a common PAMsequence. 60. The composition of embodiment 59, comprising a thirdprogrammable nuclease population, wherein none of the first programmablenuclease population, the second programmable nuclease population, andthe third programmable nuclease population recognize a common PAMsequence. 61. The composition of any one of embodiments 59-60,comprising a fourth programmable nuclease population, wherein none ofthe first programmable nuclease population, the second programmablenuclease population, the third programmable nuclease population, and thefourth programmable nuclease population recognize a common PAM sequence.62. The composition of any one of embodiments 59-61, wherein the firstprogrammable nuclease, the second programmable nuclease, or acombination thereof comprises a Cas12 protein, a Cas13 protein, or aCas14 protein. 63. The composition of any one of embodiments 59-62,wherein the third programmable nuclease comprises a Cas12 protein, aCas13 protein, or a Cas14 protein. 64. The composition of any one ofembodiments 59-63, wherein the fourth programmable nuclease comprises aCas12 protein, a Cas13 protein, or a Cas14 protein. 65. A method forcleaving a unique site of a nucleic acid molecule, comprising designinga guide nucleic acid to cleave the unique site of the nucleic acidmolecule and contacting the guide nucleic acid to a programmablenuclease and to the unique site of the nucleic acid molecule, therebycleaving the unique site of the nucleic acid molecule. 66. The method ofany one of embodiments 65-65, wherein a PAM sequence is not consideredin the designing of the guide nucleic acid. 67. The method of any one ofembodiments 65-66, wherein the programmable nuclease comprises a Casprotein. 68. The method of any one of embodiments 65-67, wherein the Casprotein is Cas14. 69. A method of sequence specific cleavage of anucleic acid molecule in a sample comprising contacting to a first PAMindependent nuclease to a flank on one side of a cleavage site thenucleic acid molecule and a second PAM independent nuclease to a flankon the other side of the cleavage site of the nucleic acid molecule. 70.The method of any one of embodiments 65-69, further comprisingcontacting the sample to a DNA fragment for sequence specific breakrepair. 71. The method of any one of embodiments 65-70, wherein the PAMindependent nuclease is a Cas protein. 72. The method of any one ofembodiments 65-71, wherein the Cas protein is a nickase. 73. The methodof any one of embodiments 65-72, wherein the Cas protein is Cas14. 74. Amethod of detecting a presence or an absence of a target nucleic acid ina sample, the method comprising: contacting a first volume to a secondvolume, wherein the first volume comprises the sample and the secondvolume comprises: i) a guide nucleic acid having at least 10 nucleotidesreverse complementary to a target nucleic acid in the sample; and ii) aprogrammable nuclease activated upon binding of the guide nucleic acidto the target nucleic acid; iii) a reporter comprising a nucleic acidand a detection moiety, wherein the second volume is at least 4-foldgreater than the first volume; and detecting the presence or the absenceof the target nucleic acid by measuring a signal produced by cleavage ofthe nucleic acid of the reporter, wherein cleavage occurs when theprogrammable nuclease is activated. 75. The method of any one ofembodiments 65-74, wherein the first volume comprises from 1 μL to 10μL. 76. The method of any one of embodiments 65-75, wherein the firstvolume comprises from 1 μL to 5 μL. 77. The method of any one ofembodiments 65-76, wherein the first volume comprises about 2 μL. 78.The method of any one of embodiments 65-77, wherein the first volumecomprises about 4 μL. 79. The method of any one of embodiments 65-78,wherein the second volume comprises from 5 μL to 40 μL. 80. The methodof any one of embodiments 65-79, wherein the second volume comprisesfrom 10 μL to 30 μL. 81. The method of any one of embodiments 65-80,wherein the second volume comprises about 20 μL. 82. The method of anyone of embodiments 65-81, wherein the second volume comprises about 30μL. 83. The method of any one of embodiments 65-82, wherein the firstvolume comprises one or more of a buffer for cell lysis, a buffer foramplification, a primer, a polymerase, target nucleic acid, a non-targetnucleic acid, a single-stranded DNA, a double-stranded DNA, a salt, abuffering agent, an NTP, a dNTP, or any combination thereof. 84. Themethod of any one of embodiments 65-83, wherein the sample is abiological sample comprising blood, serum, plasma, saliva, urine,mucosal sample, peritoneal sample, cerebrospinal fluid, gastricsecretions, nasal secretions, sputum, pharyngeal exudates, urethral orvaginal secretions, an exudate, an effusion, or tissue. 85. The methodof any one of embodiments 65-84, wherein the programmable nuclease is aprogrammable Type V CRISPR/Cas enzyme. 86. The method of any one ofembodiments 65-85, wherein the programmable Type V CRISPR/Cas enzyme isa programmable Cas12 nuclease. 87. The method of any one of embodiments65-86, wherein the programmable Cas12 nuclease is Cas12a, Cas12b,Cas12c, Cas12d, or Cas12e. 88. The method of any one of embodiments65-87, wherein the programmable Type V CRISPR/Cas enzyme is aprogrammable Cas14 nuclease. 89. The method of any one of embodiments65-88, wherein the programmable Cas14 nuclease is Cas14a, Cas14b,Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, or Cas14h. 90. The method of anyone of embodiments 65-89, wherein the programmable nuclease is aprogrammable Type VI CRISPR/Cas enzyme. 91. The method of any one ofembodiments 65-90, wherein the programmable Type VI CRISPR/Cas enzyme isa programmable Cas13 nuclease. 92. The method of any one of embodiments65-91, wherein the programmable Cas13 nuclease is Cas13a, Cas13b,Cas13c, Cas13d, or Cas13e. 93. A method of designing a plurality ofprimers for amplification of a target nucleic acid, the methodcomprising: providing a target nucleic acid, wherein a guide nucleicacid hybridizes to the target nucleic acid and wherein at least 60% of asequence of the target nucleic acid is between an F1c region and a B1region or between an F1 and a B1c region; and designing the plurality ofprimers comprising: i) a forward inner primer comprising a sequence ofthe F1c region 5′ of a sequence of an F2 region; ii) a backward innerprimer comprising a sequence of the B1c region 5′ of a sequence of a B2region; iii) a forward outer primer comprising a sequence of an F3region; and iv) a backward outer primer comprising a sequence of a B3region. 94. A method of detecting a target nucleic acid in a sample, themethod comprising: contacting the sample to: a plurality of primerscomprising: i) a forward inner primer comprising a sequencecorresponding to an F1c region 5′ of a sequence corresponding to an F2region; ii) a backward inner primer comprising a sequence correspondingto a B1c region 5′ of a sequence corresponding to a B2 region; iii) aforward outer primer comprising a sequence corresponding to an F3region; and iv) a backward outer primer comprising a sequencecorresponding to a B3 region; a guide nucleic acid, wherein the guidenucleic acid hybridizes to the target nucleic acid and wherein at least60% of a sequence of the target nucleic acid is between the F1c regionand a B1 region or between an F1 region and the B1c region; a reporter;and a programmable nuclease that cleaves the reporter when complexedwith the guide nucleic acid upon hybridization of the guide nucleic acidto the target nucleic acid; and measuring a detectable signal producedby cleavage of the reporter, wherein the measuring provides fordetection of the target nucleic acid in the sample. 95. The method ofany one of embodiments 65-94, wherein the sequence between the F1cregion and the B1 region or the sequence between the B1c region and theF1 region is at least 50% reverse complementary to the guide nucleicacid sequence. 96. The method of any one of embodiments 65-95, whereinthe guide nucleic acid sequence is reverse complementary to no more than50% of the forward inner primer, the backward inner primer, or acombination thereof. 97. The method of any one of embodiments 65-96,wherein the guide nucleic acid does not hybridize to the forward innerprimer and the backward inner primer. 98. The method of any one ofembodiments 65-97, wherein a protospacer adjacent motif (PAM) or aprotospacer flanking site (PFS) is 3′ of the target nucleic acid. 99.The method of any one of embodiments 65-98, wherein a protospaceradjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of theB1 region and 5′ of the F1c region or the protospacer adjacent motif(PAM) or a protospacer flanking site (PFS) is 3′ of the F1 region and 5′of the B1c region. 100. The method of any one of embodiments 65-99,wherein the 3′ end of the target nucleic acid is 5′ of the 5′ end of theF3c region or the 3′ end of the target nucleic acid is 5′ of the 5′ endof the B3c region. 101. The method of any one of embodiments 65-100,wherein the 3′ end of the target nucleic acid is 5′ of the 5′ end of theF2c region or 3′ end of the target nucleic acid is 5′ of the 5′ end ofthe B2c region. 102. The method of any one of embodiments 65-101,wherein the target nucleic acid is between the F1c region and the B1region and the 3′ end of the target nucleic acid is 5′ of the 3′ end ofthe F2c region, or wherein the target nucleic acid is between the B1cregion and the F1 region and the 3′ end of the target nucleic acid is 5′of the 3′ end of the B2c region. 103. The method of any one ofembodiments 65-102, wherein the guide nucleic acid has a sequencereverse complementary to no more than 50% of the forward inner primer,the backward inner primer, the forward outer primer, the backward outerprimer, or any combination thereof 104. The method of any one ofembodiments 65-103, wherein the guide nucleic acid sequence does nothybridize to the forward inner primer, the backward inner primer, theforward outer primer, the backward outer primer, or any combinationthereof. 105. The method of any one of embodiments 65-104, wherein theguide nucleic acid sequence has a sequence reverse complementary to nomore than 50% of a sequence of an F3c region, an F2c region, the F1cregion, the B1c region, an B2c region, an B3c region, or any combinationthereof. 106. The method of any one of embodiments 65-105, wherein theguide nucleic acid sequence does not hybridize to a sequence of an F3cregion, an F2c region, the F1c region, the B1c region, an B2c region, anB3c region, or any combination thereof 107. A method of designing aplurality of primer for amplification of a target nucleic acid, themethod comprising: providing the target nucleic acid comprising asequence between a B2 region and a B1 region or between an F2 region andan F1 region that hybridizes to a guide nucleic acid; and designing theplurality of primers comprising: i) a forward inner primer comprising asequence of the F1c region 5′ of a sequence of an F2 region; ii) abackward inner primer comprising a sequence of the B1c region 5′ of asequence of a B2 region; iii) a forward outer primer comprising asequence of an F3 region; and iv) a backward outer primer comprising asequence of a B3 region. 108. A method of designing a plurality ofprimer for amplification of a target nucleic acid, the methodcomprising: providing the target nucleic acid comprising a sequencebetween a F1c region and an F2c region or between a B1c region and a B2cregion that hybridizes to a guide nucleic acid; and designing theplurality of primers comprising: i) a forward inner primer comprising asequence of the F1c region 5′ of a sequence of an F2 region; ii) abackward inner primer comprising a sequence of the B1c region 5′ of asequence of a B2 region; iii) a forward outer primer comprising asequence of an F3 region; and iv) a backward outer primer comprising asequence of a B3 region. 109. A method of detecting a target nucleicacid in a sample, the method comprising: contacting the sample to: aplurality of primers comprising: i) a forward inner primer comprising asequence corresponding to an F1c region 5′ of a sequence correspondingto an F2 region; ii) a backward inner primer comprising a sequencecorresponding to a B1c region 5′ of a sequence corresponding to a B2region; iii) a forward outer primer comprising a sequence correspondingto an F3 region; and iv) a backward outer primer comprising a sequencecorresponding to a B3 region; a guide nucleic acid, wherein the targetnucleic acid comprises a sequence between a B2 region and a B1 region orbetween the F2 region and an F1 region that hybridizes to the guidenucleic acid; a reporter; and a programmable nuclease that cleaves thereporter when complexed with the guide nucleic acid upon hybridizationof the guide nucleic acid to the target nucleic acid; and measuring adetectable signal produced by cleavage of the reporter, wherein themeasuring provides for detection of the target nucleic acid in thesample. 110. A method of detecting a target nucleic acid in a sample,the method comprising: contacting the sample to: a plurality of primerscomprising: i) a forward inner primer comprising a sequencecorresponding to an F1c region 5′ of a sequence corresponding to an F2region; ii) a backward inner primer comprising a sequence correspondingto a B1c region 5′ of a sequence corresponding to a B2 region; iii) aforward outer primer comprising a sequence corresponding to an F3region; and iv) a backward outer primer comprising a sequencecorresponding to a B3 region; a guide nucleic acid, wherein the targetnucleic acid comprises a sequence between the F1c region and an F2cregion or between the B1c region and a B2c region that hybridizes to theguide nucleic acid; a reporter; and a programmable nuclease that cleavesthe reporter when complexed with the guide nucleic acid uponhybridization of the guide nucleic acid to the target nucleic acid; andmeasuring a detectable signal produced by cleavage of the reporter,wherein the measuring provides for detection of the target nucleic acidin the sample. 111. The method of any one of embodiments 65-110, whereina protospacer adjacent motif (PAM) or a protospacer flanking site (PFS)is 3′ of the B2 region and 5′ of the B1 region or the protospaceradjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of theF2 region and 5′ of the F1 region. 112. The method of any one ofembodiments 65-111, wherein a protospacer adjacent motif (PAM) or aprotospacer flanking site (PFS) is 3′ of the B1c region and 5′ of theB2c region or the protospacer adjacent motif (PAM) or a protospacerflanking site (PFS) is 3′ of the F1c region and 5′ of the F2c region.113. The method of any one of embodiments 65-112, wherein a protospaceradjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of thetarget nucleic acid. 114. The method of any one of embodiments 65-113,wherein the PAM and the PFS are 5′ of the 5′ end of the F1c region, 5′of the 5′ end of the B1c region, 3′ of the 3′ end of the F3 region, 3′of the 3′ end of the B3 region, 3′ of the 3′ end of the F2 region, 3′ ofthe 3′ end of the B2 region, or any combination thereof 115. The methodof any one of embodiments 65-114, wherein the PAM and the PFS do notoverlap the F2 region, the B3 region, the F1c region, the F2 region, theB1c region, the B2 region, or any combination thereof 116. The method ofany one of embodiments 65-115, wherein the PAM and the PFS do nothybridize to the forward inner primer, the backward inner primer, theforward outer primer, the backward outer primer, or any combinationthereof. 117. The method of any one of embodiments 65-116, wherein theplurality of primers further comprises a loop forward primer. 118. Themethod of any one of embodiments 65-117, wherein the plurality ofprimers further comprises a loop backward primer. 119. The method of anyone of embodiments 65-118, wherein the loop forward primer is between anF1c region and an F2c region. 120. The method of any one of embodiments65-119, wherein the loop backward primer is between a B1c region and aB2c region. 121. The method of any one of embodiments 65-120, whereinthe target nucleic acid comprises a single nucleotide polymorphism(SNP). 122. The method of any one of embodiments 65-121, wherein thesingle nucleotide polymorphism (SNP) comprises a HERC2 SNP, an ALDH2SNP, an EGFR SNP, a PNPLA3 SNP, a CYP2C19*2 SNP, a PAH SNP, a CFTR SNP,a 8-globin SNP, a DMD SNP, a APOB SNP, a LDLR SNP, a LDLRAP1 SNP, aPCSK9 SNP, a NF1 SNP, a PKD1 SNP, a DMPK SNP, a F9 SNP, a F8 SNP, a PKD1SNP, a PHEX SNP, or a MECP SNP. 123. The method of any one ofembodiments 65-122, wherein the single nucleotide polymorphism (SNP) isassociated with an increased risk or decreased risk of cancer. 124. Themethod of any one of embodiments 65-123, wherein the target nucleic acidcomprises a single nucleotide polymorphism (SNP), and wherein thedetectable signal is higher in the presence of a guide nucleic acid thatis 100% complementary to the target nucleic acid comprising the singlenucleotide polymorphism (SNP) than in the presence of a guide nucleicacid that is less than 100% complementary to the target nucleic acidcomprising the single nucleotide polymorphism (SNP). 125. The method ofany one of embodiments 65-124, wherein the plurality of primers and theguide nucleic acid are present together in a sample comprising thetarget nucleic acid. 126. The method of any one of embodiments 65-125,wherein the amplifying and the contacting the sample to the guidenucleic acid occurs at the same time. 127. The method of any one ofembodiments 65-126, wherein the amplifying and the contacting the sampleto the guide nucleic acid occur at different times. 128. The method ofany one of embodiments 65-127, wherein the method further comprisesproviding a polymerase, a dATP, a dTTP, a dGTP, a dCTP, or anycombination thereof 129. A method of assaying for a target nucleic acidin a sample, comprising: contacting the sample to a complex comprising aguide nucleic acid comprising a segment that is reverse complementary toa segment of the target nucleic acid and a programmable nuclease thatexhibits sequence independent cleavage upon forming a complex comprisingthe segment of the guide nucleic acid binding to the segment of thetarget nucleic acid, wherein the sample comprises at least one nucleicacid comprising at least 50% sequence identity to the segment of thetarget nucleic acid; and assaying for cleavage of at least one detectornucleic acids of a population of detector nucleic acids, wherein thecleavage indicates a presence of the target nucleic acid in the sampleand wherein absence of the cleavage indicates an absence of the targetnucleic acid in the sample. 130. The method of embodiment 129, whereinthe target nucleic acid is from 0.05% to 20% of total nucleic acids inthe sample. 131. The method of any one of embodiments 129-130, whereinthe target nucleic acid is from 0.1% to 10% of total nucleic acids inthe sample. 132. The method of any one of embodiments 129-131, whereinthe target nucleic acid is from 0.1% to 5% of total nucleic acids in thesample. 133. The method of any one of embodiments 129-132, wherein thecontacting is performed in a buffer comprising heparin and NaCl. 134.The method of any one of embodiments 129-133, wherein the NaCl is from50 mM NaCl to 200 mM NaCl. 135. The method of any one of embodiments129-134, wherein the NaCl is 100 mM NaCl. 136. The method of any one ofembodiments 129-135, wherein the heparin is from 20 μg/ml heparin to 100μg/ml heparin. 137. The method any one of embodiments 129-136, whereinthe heparin is 50 μg/ml heparin. 138. The method of any one ofembodiments 129-137, wherein the sample comprises at least one nucleicacid comprising at least 80% sequence identity to the segment of thetarget nucleic acid. 139. The method of any one of embodiments 129-138,wherein the sample comprises at least one nucleic acid comprising atleast 90% sequence identity to the segment of the target nucleic acid.140. The method of any one of embodiments 129-139, wherein the samplecomprises at least one nucleic acid comprising at least 99% sequenceidentity to the segment of the target nucleic acid. 141. The method ofany one of embodiments 129-140, wherein the sample comprises at leastone nucleic acid comprising less than 100% sequence identity to thesegment of the target nucleic acid and no less than 50% sequenceidentity to the segment of the target nucleic acid. 142. The method ofany one of embodiments 129-141, wherein the sample comprises at leastone nucleic acid comprising less than 100% sequence identity to thesegment of the target nucleic acid and no less than 80% sequenceidentity to the segment of the target nucleic acid. 143. The method ofany one of embodiments 129-142, wherein the sample comprises at leastone nucleic acid comprising less than 100% sequence identity to thesegment of the target nucleic acid and no less than 90% sequenceidentity to the segment of the target nucleic acid. 144. The method ofany one of embodiments 129-143, wherein the target nucleic acidcomprises a single nucleotide mutation. 145. The method of any one ofembodiments 129-144, wherein the segment of the target nucleic acidcomprises a single nucleotide mutation. 146. The method of any one ofembodiments 129-145, wherein the single nucleotide mutation is asynonymous substitution or a nonsynonymous substitution. 147. The methodof any one of embodiments 129-146, wherein the synonymous substitutionis a silent substitution. 148. The method of any one of embodiments129-147, wherein the nonsynonymous substitution is a missensesubstitution or a nonsense point mutation. 149. The method of any one ofembodiments 129-148, wherein the target nucleic acid comprises adeletion. 150. The method of any one of embodiments 129-149, wherein thesegment of the target nucleic acid comprises a deletion. 151. The methodof any one of embodiments 129-150, wherein the deletion comprises adeletion of from 1 to 50 nucleotides. 152. The method of any one ofembodiments 129-151, wherein the deletion comprises a deletion of from 9to 21 nucleotides. 153. The method of any one of embodiments 129-152,further comprising amplifying the target nucleic acid segment using aprimer having a region that is reverse complementary to the targetnucleic acid segment and a region that has a PAM sequence reversecomplement, thereby generating a PAM target nucleic acid having a PAMsequence adjacent to target sequence of an amplification product beforethe contacting. 154. The method of any one of embodiments 129-153,wherein the primer is a forward primer comprising the sequence encodingthe PAM and has 1-8 nucleotides from the 3′ end of the sequence encodingthe PAM. 155. The method of any one of embodiments 129-154, wherein theprimer is a forward primer comprising the sequence encoding the PAM andhas 4 nucleotides from the 3′ end of the sequence encoding the PAM. 156.The method of any one of embodiments 129-155, wherein the primer is aforward primer comprising the sequence encoding the PAM and has 5nucleotides from the 3′ end of the sequence encoding the PAM. 157. Themethod of any one of embodiments 129-156, wherein the primer is aforward primer comprising the sequence encoding the PAM and has 6nucleotides from the 3′ end of the sequence encoding the PAM. 158. Themethod of any one of embodiments 129-157, wherein the segment of thetarget nucleic acid comprises the single nucleotide mutation at 5-9nucleotides downstream of the 5′ end the segment of the target nucleicacid comprising the sequence the encoding the PAM. 159. The method ofany one of embodiments 129-158, wherein the segment of the targetnucleic acid comprises the single nucleotide mutation at 6 nucleotidesdownstream of the 5′ end the segment of the target nucleic acidcomprising the sequence the encoding the PAM. 160. The method of any oneof embodiments 129-159, wherein the segment of the target nucleic acidcomprises the single nucleotide mutation at 7 nucleotides downstream ofthe 5′ end the segment of the target nucleic acid comprising thesequence the encoding the PAM. 161. The method of any one of embodiments129-160, wherein the segment of the target nucleic acid comprises thesingle nucleotide mutation at 8 nucleotides downstream of the 5′ end thesegment of the target nucleic acid comprising the sequence the encodingthe PAM. 162. The method of any one of embodiments 129-161, wherein thesegment of the target nucleic acid comprises the deletion at 5-9nucleotides downstream of the 5′ end the segment of the target nucleicacid comprising the sequence the encoding the PAM. 163. The method ofany one of embodiments 129-162, wherein the segment of the targetnucleic acid comprises the deletion at 6 nucleotides downstream of the5′ end the segment of the target nucleic acid comprising the sequencethe encoding the PAM. 164. The method of any one of embodiments 129-163,wherein the segment of the target nucleic acid comprises the deletion at7 nucleotides downstream of the 5′ end the segment of the target nucleicacid comprising the sequence the encoding the PAM. 165. The method ofany one of embodiments 129-164, wherein the segment of the targetnucleic acid comprises the deletion at 8 nucleotides downstream of the5′ end the segment of the target nucleic acid comprising the sequencethe encoding the PAM. 166. The method of any one of embodiments 129-165,further comprising amplifying the target nucleic acid before thecontacting. 167. The method of any one of embodiments 129-166, whereinthe amplifying the target nucleic acid before the contacting comprisesusing a blocking primer. 168. The method of any one of embodiments129-167, wherein the blocking primer binds to a nucleic acid comprisingencoding the wild type sequence of the target nucleic acid segment. 169.The method of any one of embodiments 129-168, wherein the amplifyingcomprises COLD-PCR. 170. The method of any one of embodiments 129-169,wherein the COLD-PCR comprises full COLD-PCR. 171. The method of any oneof embodiments 129-170, wherein the COLD-PCR comprises fast COLD-PCR.172. The method of any one of embodiments 129-171, wherein theamplifying comprises fast COLD-PCR. 173. The method of any one ofembodiments 129-172, wherein the amplifying comprises allele-specificPCR. 174. The method of any one of embodiments 129-173, wherein theamplifying further comprises COLD-PCR. 175. The method of any one ofembodiments 129-174, further comprising removing a nucleic acidcomprising at least 50% sequence identity to the target nucleic acid bybinding a protein to the nucleic acid before the contacting. 176. Themethod of any one of embodiments 129-175, wherein the protein is anantibody. 177. The method of any one of embodiments 129-176, wherein theprotein is a programmable nuclease without endonuclease activity. 178.The method of any one of embodiments 129-177, further comprising bindinga protein to the target nucleic acid to remove other nucleic acids ofthe sample. 179. The method of any one of embodiments 129-178, whereinthe other nucleic acids comprise a nucleic acid comprising at least 50%sequence identity to the target nucleic acid. 180. The method of any oneof embodiments 129-179, wherein the protein is attached to a surface.181. The method of any one of embodiments 129-180, wherein the removingof the other nucleic acids comprises washing away nucleic acids that arenot bound to the protein. 182. The method of any one of embodiments129-181, wherein the protein is an antibody. 183. The method of any oneof embodiments 129-182, wherein the protein is a programmable nucleasewithout endonuclease activity. 184. The method of any one of embodiments129-183, wherein the programmable nuclease is a target nucleic acidactivated effector protein that exhibits sequence independent cleavageupon activation. 185. The method of any one of embodiments 129-184,wherein the programmable nuclease is an RNA guided nuclease. 186. Themethod of any one of embodiments 129-185, wherein the programmablenuclease comprises a Cas nuclease. 187. The method of any one ofembodiments 129-186, wherein the Cas nuclease is Cas13. 188. The methodof any one of embodiments 129-187, wherein the Cas13 is Cas13a, Cas13b,Cas13c, Cas13d, or Cas13e. 189. The method of any one of embodiments129-188, wherein the Cas nuclease is Cas12. 190. The method of any oneof embodiments 129-189, wherein the Cas12 is Cas12a, Cas12b, Cas12c,Cas12d, or Cas12e. 191. The method of any one of embodiments 129-190,wherein the Cas nuclease is Cas14. 192. The method of any one ofembodiments 129-191, wherein the Cas14 is Cas14a, Cas14b, Cas14c,Cas14d, Cas14e, Cas14f, Cas14g, or Cas14h. 193. The method of any one ofembodiments 129-192, wherein the Cas nuclease is Csm1, Cas9, C2c4, C2c8,C2c5, C2c10, or C2c9. 194. The method of any one of embodiments 129-193,wherein the guide nucleic acid comprises a crRNA. 195. The method of anyone of embodiments 129-194, wherein the guide nucleic acid comprises acrRNA and a tracrRNA. 196. The method of any one of embodiments 129-195,wherein cleavage of at least one detector nucleic acid yields a signal.197. The method of any one of embodiments 129-196, wherein cleavage ofat least one detector nucleic acid activates a photoexcitablefluorophore. 198. The method of any one of embodiments 129-197, whereincleavage of at least one detector nucleic acid deactivates aphotoexcitable fluorophore. 199. The method of any one of embodiments129-198, wherein the signal is present prior to detector nucleic acidcleavage. 200. The method of any one of embodiments 129-199, wherein thesignal is absent prior to detector nucleic acid cleavage. 201. Themethod of any one of embodiments 129-200, wherein the sample comprisesblood, serum, plasma, saliva, urine, mucosal sample, peritoneal sample,cerebrospinal fluid, gastric secretions, nasal secretions, sputum,pharyngeal exudates, urethral or vaginal secretions, an exudate, aneffusion, or tissue. 202. The method of any one of embodiments 129-201,wherein the single nucleotide mutation is a single nucleotidepolymorphism. 203. A method, comprising: contacting a programmablenuclease comprising a polypeptide having endonuclease activity and aguide nucleic acid to a target nucleic acid in a buffer comprisingheparin. 204. The method of any one of embodiments 129-203, wherein theheparin is present at a concentration of from 1 to 100 μg/ml heparin.205. The method of any one of embodiments 129-204, wherein the heparinis present at a concentration of from 40 to 60 μg/ml heparin. 206. Themethod of any one of embodiments 129-205, wherein the heparin is presentat a concentration 50 μg/ml heparin. 207. The method of any one ofembodiments 129-206, wherein the buffer further comprises NaCl. 208. Themethod of any one of embodiments 129-207, wherein the NaCl is present ata concentration of from 1 to 200 mM NaCl. 209. The method of any one ofembodiments 129-208, wherein the NaCl is present at a concentration offrom 80 to 120 mM NaCl. 210. The method of any one of embodiments129-209, wherein the NaCl is present at a concentration of 100 mM NaCl.211. The method of any one of any one of embodiments 129-210, whereinthe target nucleic acid is a substrate target nucleic acid. 212. Themethod of any one of embodiments 129-211, wherein the substrate nucleicacid comprises a cancer allele. 213. The method of any one ofembodiments 129-212, wherein the cancer allele is present at a lowconcentration relative to a wild type allele. 214. The method of any oneof embodiments 129-213, wherein the substrate target nucleic acidcomprises a splice variant. 215. The method of any one of embodiments129-214, wherein the substrate target nucleic acid comprises an editedbase. 216. The method of any one of embodiments 129-215, wherein thesubstrate target nucleic acid comprises a bisulfite-treated base. 217.The method of any one of embodiments 129-216, wherein the substratetarget nucleic acid comprises a segment that is reverse complementary toa segment of the guide nucleic acid. 218. A method of designing aplurality of primers for amplification of a target nucleic acid, themethod comprising: providing a target nucleic acid, wherein a guidenucleic acid hybridizes to the target nucleic acid and wherein at least60% of a sequence of the target nucleic acid is between an F1c regionand a B1 region or between an F1 and a B1c region; and designing theplurality of primers comprising: i) a forward inner primer comprising asequence of the F1c region 5′ of a sequence of an F2 region; ii) abackward inner primer comprising a sequence of the B1c region 5′ of asequence of a B2 region; iii) a forward outer primer comprising asequence of an F3 region; and iv) a backward outer primer comprising asequence of a B3 region. 219. A method of detecting a target nucleicacid in a sample, the method comprising: contacting the sample to: aplurality of primers comprising: i) a forward inner primer comprising asequence corresponding to an F1c region 5′ of a sequence correspondingto an F2 region; ii) a backward inner primer comprising a sequencecorresponding to a B1c region 5′ of a sequence corresponding to a B2region; iii) a forward outer primer comprising a sequence correspondingto an F3 region; and iv) a backward outer primer comprising a sequencecorresponding to a B3 region; a guide nucleic acid, wherein the guidenucleic acid hybridizes to the target nucleic acid and wherein at least60% of a sequence of the target nucleic acid is between the F1c regionand a B1 region or between an F1 region and the B1c region; a reporter;and a programmable nuclease that cleaves the reporter when complexedwith the guide nucleic acid upon hybridization of the guide nucleic acidto the target nucleic acid; and measuring a detectable signal producedby cleavage of the reporter, wherein the measuring provides fordetection of the target nucleic acid in the sample. 220. The method ofany one of embodiments 129-219, wherein the sequence between the F1cregion and the B1 region or the sequence between the B1c region and theF1 region is at least 50% reverse complementary to the guide nucleicacid sequence. 221. The method of any one of embodiments 129-220,wherein the guide nucleic acid sequence is reverse complementary to nomore than 50% of the forward inner primer, the backward inner primer, ora combination thereof. 222. The method of any one of embodiments129-221, wherein the guide nucleic acid does not hybridize to theforward inner primer and the backward inner primer. 223. The method ofany one of embodiments 129-222, wherein a protospacer adjacent motif(PAM) or a protospacer flanking site (PFS) is 3′ of the target nucleicacid. 224. The method of any one of embodiments 129-223, wherein aprotospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is3′ of the B1 region and 5′ of the F1c region or the protospacer adjacentmotif (PAM) or a protospacer flanking site (PFS) is 3′ of the F1 regionand 5′ of the B1c region. 225. The method of any one of embodiments129-224, wherein the 3′ end of the target nucleic acid is 5′ of the 5′end of the F3c region or the 3′ end of the target nucleic acid is 5′ ofthe 5′ end of the B3c region. 226. The method of any one of embodiments129-225, wherein the 3′ end of the target nucleic acid is 5′ of the 5′end of the F2c region or 3′ end of the target nucleic acid is 5′ of the5′ end of the B2c region. 227. The method of any one of embodiments129-226, wherein the target nucleic acid is between the F1c region andthe B1 region and the 3′ end of the target nucleic acid is 5′ of the 3′end of the F2c region, or wherein the target nucleic acid is between theB1c region and the F1 region and the 3′ end of the target nucleic acidis 5′ of the 3′ end of the B2c region. 228. The method of any one ofembodiments 129-227, wherein the guide nucleic acid has a sequencereverse complementary to no more than 50% of the forward inner primer,the backward inner primer, the forward outer primer, the backward outerprimer, or any combination thereof. 229. The method of any one ofembodiments 129-228, wherein the guide nucleic acid sequence does nothybridize to the forward inner primer, the backward inner primer, theforward outer primer, the backward outer primer, or any combinationthereof 230. The method of any one of embodiments 129-229, wherein theguide nucleic acid sequence has a sequence reverse complementary to nomore than 50% of a sequence of an F3c region, an F2c region, the F1cregion, the B1c region, an B2c region, an B3c region, or any combinationthereof 231. The method of any one of embodiments 129-230, wherein theguide nucleic acid sequence does not hybridize to a sequence of an F3cregion, an F2c region, the F1c region, the B1c region, an B2c region, anB3c region, or any combination thereof 232. A method of designing aplurality of primer for amplification of a target nucleic acid, themethod comprising: providing the target nucleic acid comprising asequence between a B2 region and a B1 region or between an F2 region andan F1 region that hybridizes to a guide nucleic acid; and designing theplurality of primers comprising: i) a forward inner primer comprising asequence of the F1c region 5′ of a sequence of an F2 region; ii) abackward inner primer comprising a sequence of the B1c region 5′ of asequence of a B2 region; iii) a forward outer primer comprising asequence of an F3 region; and iv) a backward outer primer comprising asequence of a B3 region. 233. A method of designing a plurality ofprimer for amplification of a target nucleic acid, the methodcomprising: providing the target nucleic acid comprising a sequencebetween a F1c region and an F2c region or between a B1c region and a B2cregion that hybridizes to a guide nucleic acid; and designing theplurality of primers comprising: i) a forward inner primer comprising asequence of the F1c region 5′ of a sequence of an F2 region; ii) abackward inner primer comprising a sequence of the B1c region 5′ of asequence of a B2 region; iii) a forward outer primer comprising asequence of an F3 region; and iv) a backward outer primer comprising asequence of a B3 region. 234. A method of detecting a target nucleicacid in a sample, the method comprising: contacting the sample to: aplurality of primers comprising: i) a forward inner primer comprising asequence corresponding to an F1c region 5′ of a sequence correspondingto an F2 region; ii) a backward inner primer comprising a sequencecorresponding to a B1c region 5′ of a sequence corresponding to a B2region; iii) a forward outer primer comprising a sequence correspondingto an F3 region; and iv) a backward outer primer comprising a sequencecorresponding to a B3 region; a guide nucleic acid, wherein the targetnucleic acid comprises a sequence between a B2 region and a B1 region orbetween the F2 region and an F1 region that hybridizes to the guidenucleic acid; a reporter; and a programmable nuclease that cleaves thereporter when complexed with the guide nucleic acid upon hybridizationof the guide nucleic acid to the target nucleic acid; and measuring adetectable signal produced by cleavage of the reporter, wherein themeasuring provides for detection of the target nucleic acid in thesample. 235. A method of detecting a target nucleic acid in a sample,the method comprising: contacting the sample to: a plurality of primerscomprising: i) a forward inner primer comprising a sequencecorresponding to an F1c region 5′ of a sequence corresponding to an F2region; ii) a backward inner primer comprising a sequence correspondingto a B1c region 5′ of a sequence corresponding to a B2 region; iii) aforward outer primer comprising a sequence corresponding to an F3region; and iv) a backward outer primer comprising a sequencecorresponding to a B3 region; a guide nucleic acid, wherein the targetnucleic acid comprises a sequence between the F1c region and an F2cregion or between the B1c region and a B2c region that hybridizes to theguide nucleic acid; a reporter; and a programmable nuclease that cleavesthe reporter when complexed with the guide nucleic acid uponhybridization of the guide nucleic acid to the target nucleic acid; andmeasuring a detectable signal produced by cleavage of the reporter,wherein the measuring provides for detection of the target nucleic acidin the sample. 236. The method of any one of embodiments 129-235,wherein a protospacer adjacent motif (PAM) or a protospacer flankingsite (PFS) is 3′ of the B2 region and 5′ of the B1 region or theprotospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is3′ of the F2 region and 5′ of the F1 region. 237. The method of any oneof embodiments 129-236, wherein a protospacer adjacent motif (PAM) or aprotospacer flanking site (PFS) is 3′ of the B1c region and 5′ of theB2c region or the protospacer adjacent motif (PAM) or a protospacerflanking site (PFS) is 3′ of the F1c region and 5′ of the F2c region.238. The method of any one of embodiments 129-237, wherein a protospaceradjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of thetarget nucleic acid. 239. The method of any one of embodiments 129-238,wherein the PAM and the PFS are 5′ of the 5′ end of the F1c region, 5′of the 5′ end of the B1c region, 3′ of the 3′ end of the F3 region, 3′of the 3′ end of the B3 region, 3′ of the 3′ end of the F2 region, 3′ ofthe 3′ end of the B2 region, or any combination thereof 240. The methodof any one of embodiments 129-239, wherein the PAM and the PFS do notoverlap the F2 region, the B3 region, the F1c region, the F2 region, theB1c region, the B2 region, or any combination thereof 241. The method ofany one of embodiments 129-240, wherein the PAM and the PFS do nothybridize to the forward inner primer, the backward inner primer, theforward outer primer, the backward outer primer, or any combinationthereof. 242. The method of any one of embodiments 129-241, wherein theplurality of primers further comprises a loop forward primer. 243. Themethod of any one of embodiments 129-242, wherein the plurality ofprimers further comprises a loop backward primer. 244. The method of anyone of embodiments 129-243, wherein the loop forward primer is betweenan F1c region and an F2c region. 245. The method of any one ofembodiments 129-244, wherein the loop backward primer is between a B1cregion and a B2c region. 246. The method of any one of embodiments129-245, wherein the target nucleic acid comprises a single nucleotidepolymorphism (SNP). 247. The method of any one of embodiments 129-246,wherein the single nucleotide polymorphism (SNP) comprises a HERC2 SNP.248. The method of any one of embodiments 129-247, wherein the singlenucleotide polymorphism (SNP) is associated with an increased risk ordecreased risk of cancer. 249. The method of any one of embodiments129-248, wherein the target nucleic acid comprises a single nucleotidepolymorphism (SNP), and wherein the detectable signal is higher in thepresence of a guide nucleic acid that is 100% complementary to thetarget nucleic acid comprising the single nucleotide polymorphism (SNP)than in the presence of a guide nucleic acid that is less than 100%complementary to the target nucleic acid comprising the singlenucleotide polymorphism (SNP). 250. The method of any one of embodiments129-249, wherein the plurality of primers and the guide nucleic acid arepresent together in a sample comprising the target nucleic acid. 251.The method of any one of embodiments 129-250, wherein the amplifying andthe contacting the sample to the guide nucleic acid occurs at the sametime. 252. The method of any one of embodiments 129-251, wherein theamplifying and the contacting the sample to the guide nucleic acid occurat different times. 253. The method of any one of embodiments 129-252,wherein the method further comprises providing a polymerase, a dATP, adTTP, a dGTP, a dCTP, or any combination thereof. 254. A method ofassaying for a target nucleic acid segment in a sample, wherein thetarget nucleic acid segment lacks a PAM sequence, comprising: amplifyingthe target nucleic acid segment using a primer having a region that isreverse complementary to the target nucleic acid segment and a regionthat has a PAM sequence reverse complement, thereby generating a PAMtarget nucleic acid having a PAM sequence adjacent to a target sequenceof an amplification product; contacting the PAM target nucleic acid to aPAM-dependent sequence specific nuclease complex comprising a guidenucleic acid and a programmable nuclease that exhibits sequenceindependent cleavage upon forming a complex comprising the segment ofthe guide nucleic acid binding to the segment of the PAM target nucleicacid; and assaying for cleavage of at least one detector nucleic acid ofa population of detector nucleic acids, wherein the cleavage indicates apresence of the target nucleic acid in the sample and wherein theabsence of the cleavage indicates an absence of the target nucleic acidin the sample. 255. The method of embodiment 254, wherein the sequenceencoding the PAM comprises dUdUdUN. 256. The method any one ofembodiments 254-255, wherein the primer is a forward primer comprisingthe sequence encoding the PAM and has 3 nucleotides from the 3′ end ofthe sequence encoding the PAM. 257. The method any one of embodiments254-256, wherein the primer is a forward primer comprising the sequenceencoding the PAM and has 1-2 or 4-8 nucleotides from the 3′ end of thesequence encoding the PAM. 258. The method of any one of embodiments254-257, wherein the primer is a forward primer comprising the sequenceencoding the PAM and has 2 nucleotides from the 3′ end of the sequenceencoding the PAM. 259. The method of any one of embodiments 254-258,wherein the primer is a forward primer comprising the sequence encodingthe PAM and has 4 nucleotides from the 3′ end of the sequence encodingthe PAM. 260. The method of any one of embodiments 254-259, wherein theprimer is a forward primer comprising the sequence encoding the PAM andhas 5 nucleotides from the 3′ end of the sequence encoding the PAM. 261.The method of any one of embodiments 254-260, wherein the primer is aforward primer comprising the sequence encoding the PAM and has 6nucleotides from the 3′ end of the sequence encoding the PAM. 262. Themethod of any one of embodiments 254-261, wherein a mismatch for singlenucleotide polymorphism (SNP) detection is 3-10 nucleotides downstreamof the PAM in PAM target nucleic acid. 263. The method of any one ofembodiments 254-262, wherein a mismatch for single nucleotidepolymorphism (SNP) detection is 6 nucleotides downstream of the PAM inPAM target nucleic acid. 264. The method of any one of embodiments254-263, wherein a mismatch for single nucleotide polymorphism (SNP)detection is 7 nucleotides downstream of the PAM in PAM target nucleicacid. 265. The method of any one of embodiments 254-264, wherein amismatch for single nucleotide polymorphism (SNP) detection is 8nucleotides downstream of the PAM in PAM target nucleic acid. 266. Themethod of any one of embodiments 254-265, wherein the amplifyingcomprises thermal cycling amplification. 267. The method of any one ofembodiments 254-266, wherein the amplifying comprises isothermalamplification. 268. The method of any one of embodiments 254-267,wherein the isothermal amplification is select from the group consistingof isothermal recombinase polymerase amplification (RPA), transcriptionmediated amplification (TMA), strand displacement amplification (SDA),helicase dependent amplification (HDA), loop mediated amplification(LAMP), rolling circle amplification (RCA), single primer isothermalamplification (SPIA), ligase chain reaction (LCR), simple methodamplifying RNA targets (SMART), improved multiple displacementamplification (IMDA), and nucleic acid sequence-based amplification(NASBA). 269. The method of any one of embodiments 254-268, wherein theproducing, the contacting, and the assaying are performed in a commonreaction volume. 270. The method of any one of embodiments 254-269,wherein the programmable nuclease is a nucleic acid activated effectorprotein that exhibits sequence independent cleavage upon activation.271. The method of any one of embodiments 254-270, wherein theprogrammable nuclease is an RNA guided nuclease. 272. The method of anyone of embodiments 254-271, wherein the programmable nuclease comprisesa Cas nuclease. 273. The method of any one of embodiments 254-272,wherein the Cas nuclease is Cas12. 274. The method of any one ofembodiments 254-273, wherein the Cas12 is Cas12a, Cas12b, Cas12c,Cas12d, or Cas12e. 275. The method of any one of embodiments 254-274,wherein the cas nuclease is Cas13. 276. The method of any one ofembodiments 254-275, wherein the cas nuclease is Cas13a, Cas13b, Cas13c,or Cas13d. 277. The method of any one of embodiments 254-276, whereinthe guide nucleic acid comprises a crRNA. 278. The method of any one ofembodiments 254-277, wherein cleavage of at least one detector nucleicacid yields a signal. 279. The method of any one of embodiments 254-278,wherein cleavage of at least one detector nucleic acid activates aphotoexcitable fluorophore. 280. The method of any one of embodiments254-279, wherein cleavage of at least one detector nucleic aciddeactivates a photoexcitable fluorophore. 281. The method of any one ofembodiments 254-280, wherein the signal is present prior to detectornucleic acid cleavage. 282. The method of any one of embodiments254-281, wherein the signal is absent prior to detector nucleic acidcleavage. 283. The method of any one of embodiments 254-282, wherein theat least one detector nucleic acid comprises a nucleic acid comprising adetectable moiety. 284. The method of any one of embodiments 254-283,wherein the at least one detector nucleic acid comprises a nucleic acidcomprising at least two nucleotides, a fluorophore, and a fluorescencequencher, wherein the fluorophore and the fluorescence quencher arelinked by the nucleic acid. 285. The method of any one of embodiments254-284, wherein the sample comprises blood, serum, plasma, saliva,urine, mucosal sample, peritoneal sample, cerebrospinal fluid, gastricsecretions, nasal secretions, sputum, pharyngeal exudates, urethral orvaginal secretions, an exudate, an effusion, or tissue. 286. The methodof any one of embodiments 254-285, wherein the target nucleic acidcomprises a sequence encoding a single nucleotide polymorphism (SNP).287. The method of any one of embodiments 254-286, wherein the targetnucleic acid comprises a sequence encoding a wild type sequence. 288.The method of any one of embodiments 254-287, wherein the SNP is in theEGFR gene. 289. The method of any one of embodiments 254-288, whereinthe SNP is associated with a disease. 290. The method of any one ofembodiments 254-289, wherein the SNP is a HERC2 SNP, an ALDH2 SNP, anEGFR SNP, a PNPLA3 SNP, a CYP2C19*2 SNP, a PAH SNP, a CFTR SNP, aβ-globin SNP, a DMD SNP, a APOB SNP, a LDLR SNP, a LDLRAP1 SNP, a PCSK9SNP, a NF1 SNP, a PKD1 SNP, a DMPK SNP, a F9 SNP, a F8 SNP, a PKD1 SNP,a PHEX SNP, or a MECP SNP. 291. The method of any one of embodiments254-290, wherein the disease is cancer. 292. The method of any one ofembodiments 254-291, wherein the disease is a genetic disorder. 293. Themethod of any one of embodiments 254-292, wherein the SNP is associatedwith altered phenotype compared to a wild type sequence.

EXAMPLES

The following examples are illustrative and non-limiting to the scope ofthe devices, systems, fluidic devices, kits, and methods describedherein.

Example 1 Substrate Screen (Trans Cleavage)

Trans cleavage assays were performed activity buffer (buffer: 120 mMNaCl, 5 mM MgCl₂, 20 mM Tris pH 7.5, 1% glycerol). A final concentrationof 100 nM and 50 nM of different target dsDNA (varying in PAM andmismatches) and ssDNA-FQ reporter molecule were used in the assayrespectively. Target dsDNA was obtained by annealing complementary ssDNAprimers with 2:1 ratio of non-target strand to target strand inhybridization buffer (10× Hybridization buffer: 500 mM NaCl, 10 mM TrispH 8.0, 1 mM EDTA) This ensures double-stranded DNA is being detectedinstead of single-stranded DNA.

crRNA was synthesized via in-vitro transcription using T7 RNA polymeraseand a DNA template that consists of T7 binding site sequence followed byrepeat region and targets sequence. Synthesized crRNA was then purifiedvia bead purification and quantified using Quant-It miRNA kit.

To prepare the assay, a mastermix that consists of nuclease free water,ssDNA-FQ reporter, and 5× activity buffer was made and distributed to 12different 1.5 mL microcentrifuge tubes. Each tube is for each proteinortholog. Add the protein of interest and the guide RNA to each tube andproceed to incubation for 20 minutes at 37 C. Transfer 16 uL of each ofthe incubated mastermix per reaction.

To activate the assay, add 4 uL of 500 nM target dsDNA into thereaction. Place the plate to a fluorescence reader for 2 hours.

Example 2 Cis (Target) Cleavage Assays

Cis (target) cleavage assays were performed at 25° C. or 37° C. inactivity buffer (120 mM NaCl, 5 mM MgCl₂, 20 mM Tris pH 7.5, 1%glycerol). Cas12a-crRNA complex formation was performed in activitybuffer, generally at a molar ratio of 1:1.25 protein to crRNA at 37° C.for 10 min, a target dsDNA. The target for cis-cleavage is a PCR productthat is 1200 bp long and contains the target sequence at the 700thposition. A restriction site for BamHI was also introduced around thevicinity of the target sequence. Unless otherwise indicated, finalconcentrations of protein, guide and targets were 100 nM, 125 nM and 15nM, respectively, for all reactions. Reactions were quenched with 6×loading dye and resolved by prestained 2% agarose gel (1×TAE buffer).The cis cleavage reaction has the same conditions as the trans cleavagereaction but without a reporter molecule and a target dsDNA finalconcentration of 15 nM.

Example 3 Limit of Detection Assays

To monitor the limit of detection (LOD) of each chosen protein ortholog,DETECTR assay was used. The reaction cocktail is identical to that ofthe substrate screen assay. The only difference is the targetconcentration; target concentrations of 100 nM, 10 nM, 1 nM, 100 pM, 10pM, 1 pM, 100 fM, 10 fM, and 1 fM were prepared via rehybridization asmentioned above and serial dilution.

Example 4 Guide Processing Assays

Pre-crRNA cleavage assays are performed at 37° C. in Activity Bufferbased on previous buffer optimization experiments 100-fold molar excessof Cpf1 relative to synthesized crRNA (final concentrations of 100 nMand <1 nM, respectively). Unless otherwise indicated, the reaction isquenched after 1 h with 2×RNA loading dye (100% formamide, 0.025% (w/v)bromophenol blue and 200 μg/mL heparin). After quenching, reactions aredenatured at 95° C. for 2 min before resolving by 15% denaturing PAGE(1×TBE buffer).

Example 5 Temperature Assays

Each protein is pre-complexed by adding its crRNA and preincubated at 25C for 1 hour. After the 1 hour period, each protein complex is incubatedin different temperatures for 10 minutes. The temperatures are: 4, 22,37, and 48. After incubation, the protein complex is tested via DETECTR.

Example 6 Nickase Assays

A pUC19 is treated with a Cas14 comprising a guide nucleic comprising asegment of nucleic acid that is reverse complementary with a segment ofpUC19. After treatment, a band is produced when run on a gel that ishigher than the linearized pUC19 fragment produced by digestion withEcoR1. A band that is higher than the linearized pUC19 is produced whenno tracr nucleic acid is added to the treatment, and a band that ishigher than the linearized pUC19 is produced when either a tracr nucleicacid comprising or lacking a PAM sequence is added to the treatment.This indicates that the Cas14 is a nickase and is PAM independent andtracr nucleic acid independent. However, a lower band than thelinearized pUC19 is produced when no guide nucleic acid is added,indicating that the cleavage is guide nucleic acid directed.

Example 7 Optimization of Temperature and Temperature Tolerance ofCRISPR-Cas Proteins in CRISPR Diagnostics

This example describes optimization of temperature and temperaturetolerance of CRISPR-Cas proteins in CRISPR diagnostics. The CRISPRdiagnostics of the present disclosure leverage the unique biochemicalproperties of Type V (e.g., Cas12) and Type VI (e.g., Cas13) CRISPR-Casproteins to enable the specific detection of nucleic acids. Theseproteins are directed to their target nucleic acid by a CRISPR RNA(crRNA), which is also known as a guide RNA (gRNA). Once bound to acomplementary target sequence, the Cas protein initiates indiscriminatecleavage of surrounding single-strand DNA or single-strand RNA. Whencoupled to a quenched fluorescence reporter or other cleavage reporter,fluorescent or other signal can be generated by the Cas protein only inthe presence of the target nucleic acid. CRISPR-Cas proteins have beenisolated from a variety of natural contexts and therefore have differenttolerances for elevated temperatures and optimal temperature ranges.These different tolerances for temperature can be used to activate orinhibit the proteins at different stages to allow for other molecularprocesses, such as target amplification, to occur.

A Cas12 variant (SEQ ID NO: 11), LbCas12a (SEQ ID NO: 1), and LbuCas13a(SEQ ID NO: 104) were incubated at 25° C., 30° C., 35° C., 40° C., 45°C., and 50° C. with a target nucleic acid sequence. Detection assaysusing the various Cas proteins were set up using 1 nM DNA target forCas12 proteins and 25 μM RNA target for Cas13a. The max_rate(fluriescence units/2 min) was determined for evaluating the efficiencyof the proteins at various temperatures. Darker squares indicate ahigher max_rate and more efficient activity.

FIG. 18 shows activity of three programmable nucleases, a Cas12 variant(SEQ ID NO: 11), LbCas12a (SEQ ID NO: 1), and LbuCas13a (SEQ ID NO: 104,also referred to herein as Lbu C2C2). The results show that thefunctional range for the Cas12 variant (SEQ ID NO: 11) is between 25° C.and 45° C., with maximal activity at 35° C. For the Type V Cas12 proteinLbCas12a (SEQ ID NO: 1) the functional range is from 35° C. to 50° C.with peak activity around 40° C. For the Type VI protein LbuCas13a (SEQID NO: 104) the functional range is between 25° C. and 40° C. withmaximal activity between 30° C. and 35° C. As suggested in FIG. 18, itappears that Type V proteins, such as the Cas12 variant (SEQ ID NO: 11)and LbCas12a (SEQ ID NO: 1), may be stable and functional at elevatedtemperatures. To test how stable each of these proteins are, proteinswere incubated for 15 minutes at 45° C., 50° C., 55° C., 60° C., 65° C.,or 70° C. and then decreased the reaction temperature to 37° C.

FIG. 19 shows the results of incubating two Cas12 proteins, SEQ ID NO: 1and SEQ ID NO: 11, for 15 minutes at 45° C., 50° C., 55° C., 60° C., 65°C., or 70° C. and then decreasing the reaction temperature to 37° C.LbCas12a (SEQ ID NO: 1) was found to be functional even after incubationat 65° C. The Cas12 variant (SEQ ID NO: 11) was found to have noactivity while at temperatures above 50° C., but after lowering thetemperature to 37° C., the enzymatic activity of the protein returned.This temperature shifting may be exploitable for use in isothermalamplification methods, where the amplification occurs at a highertemperature, but after lowering the reaction temperature the Cas proteincan be activated without compromising its functionality.

FIG. 20 shows that the stability of the Cas12 variant (SEQ ID NO: 11) atelevated temperatures is dependent on the buffer composition. Stabilityof the Cas12 variant was assessed after exposure to elevatedtemperatures for 30 minutes and then lowering the reaction temperatureto 37° C. A variety of buffers were tested to determine their impact onthe ability to turn the Cas12 variant on and off based on the reactiontemperature. 0.5× NEBuffer4 (New England Biolabs, 1×: 50 mM PotassiumAcetate; 20 mM Tris-acetate, pH 7.9; 10 mM Magnesium Acetate; 1 mMDTT)+0.05% Tween gave the best results, followed by 1× MBuffer3 (20 mMHEPES pH 7.5; 2 mM Potassium Acetate; 5 mM Mg Acetate; 1% glycerol;0.00016% Triton-X). 0.5× of Isothermal Amplification (IsoAmp) buffer(New England Biolabs) inhibited the Cas12 variant reaction completely.

Example 8 Optimization of Assay Conditions for CRISPR DETECTR-BasedDiagnostic Assays

This example describes optimization of assay conditions for theCRISPR-Cas DETECTR-based diagnostic assays disclosed herein. Thecomponents of the DETECTR reaction, such as protein concentration,crRNA, and buffer components impact the rate and efficiency of thereaction. Optimization of the buffers allows for the development of anassay with increased sensitivity and specificity.

Improvements to buffers and assay conditions were identified forLbuCas13a (SEQ ID NO: 104) included 100 ng/μL of tRNA. The performanceof a HEPES pH 6.8 buffer for Cas13a detection (20 mM HEPES pH 6.8; 50 mMKCl; 5 mM MgCl₂; 10 μg/mL BSA; 100 ng/μL tRNA; 0.01% Igepal Ca-630(NP-40); 5% Glycerol) is shown on the graph is the middle-most line.Cas13a was incubated with 1 μM of target RNA at 37 C with varyingconcentrations of tRNA in the reaction buffer. As a control, the assaywas also performed with 0 μM of the target RNA. FIG. 21 shows graphs ofactivity of a Cas13 (SEQ ID NO: 104), as measured by fluorescence, with(left graph) and without (right graph) activator over time. FIG. 21shows that increasing the amount of tRNA in the reaction decreases theefficiency of the Cas13a detection assay. Similarly, decreasing theamount of tRNA in the reaction or eliminating it completely, increasesthe efficiency of the Cas13a detection assay without dramaticallychanging the stability of the reaction in the absence of activator.

Urea is an additive that is used to increase the efficiency of someenzymatic reactions, such as proteinase K digestion, and is present inurine. To evaluate inhibition of Cas13a activity in the DETECTR assays,1 μM of target RNA at 37° C. was incubated with varying concentrationsof urea. The activator, shown in the following graphs, is the targetRNA. FIG. 22 shows inhibition of Cas13a (SEQ ID NO: 104) activity by SDSand urea. FIG. 22A shows the Cas13a (SEQ ID NO: 104) detection assayperformed in the presence of 0-200 mM urea. Concentrations above 300 mMurea inhibited the assay (left graph shows with activator and rightgraph shows without activator). The orange line indicates theperformance of the assay with 0 mM urea (a control showing uninhibitedCas13a activity). SDS is a common inhibitor of RNases and is used toeliminate RNase contamination and denature proteins. To evaluateinhibition of Cas13a activity in DETECTR assays, 1 pM target RNA at 37°C. was incubated with varying amounts of SDS. FIG. 22B shows completeinhibition of Cas13a (SEQ ID NO: 104) upon addition of 0.1% or greateramounts of SDS to the reaction (left graph shows with activator andright graph shows without activator). The orange line indicatesperformance of Cas13a with 0% SDS (a control showing uninhibited Cas13aactivity).

The importance of salt type and salt concentration on the performance ofCas13a in a DETECTR assay was evaluated. DETECTR assays were performedwith 10 pM of target or 0 pM of target (control). FIG. 23 shows theperformance of Cas13a (SEQ ID NO: 104) in DETECTR reactions with varyingconcentrations of salt. FIG. 23A shows the results of varying theconcentration of NaCl in a Cas13a (SEQ ID NO: 104) DETECTR reaction.FIG. 23B shows the results of varying the concentration of KCl in aCas13a (SEQ ID NO: 104) DETECTR reaction. Cas13a performed comparablybetween NaCl and KCl salt types. Cas13a performance decreased at 30 mMsalt and below, and was inhibited by salt concentrations above 80 mM.

The importance of DTT in different salt types and its impact on Cas13a(SEQ ID NO: 104) performance in a DETECTR assay was evaluated. DTT wasused to stabilize proteins, such as RNase inhibitors, and increase theefficiency of some enzymes. DETECTR assays were carried out using Cas13afor detection of 10 pM of target or no target (control). FIG. 24 showsoptimization of DTT concentration in a Cas13a (SEQ ID NO: 104) DETECTRassay. FIG. 24A shows activity of a Cas13a (SEQ ID NO: 104) at varyingDTT concentration in NaCl. FIG. 24B shows activity of a Cas13a (SEQ IDNO: 104) at varying DTT concentrations in KCl. The orange bar indicatesbuffer conditions with 50 mM KCl and no DTT. In addition to theindicated KCl and DTT concentration, each buffer condition alsocontained 20 mM HEPES pH 6.8, 5 mM MgCl₂, 10 μg/mL BSA, 100 μg/mL tRNA,0.01% Igepal Ca-630 (NP-40), and 5% Glycerol). The results showed thatthe Cas13a DETECTR assay was not affected by DTT concentrations from0-10 mM in buffers containing either NaCl or KCl.

Reporter choice for the Cas13a DETECTR assay was evaluated. The quenchedfluorescent reporter generates the fluorescent signal that is used tomonitor Cas13a detection performance in the DETECTR assays. A variety ofdifferent RNA reporter sequences was evaluated for their impact on assayperformance. Cas13a detection assays were performed with either 1 pMtarget RNA or no target RNA at 37° C. Reactions were performed in eithera HEPES pH 6.8 Cas13a reaction buffer (HEPES pH 6.8 buffer with tRNA: 20mM HEPES pH 6.8; 50 mM KCl; 5 mM MgCl₂; 10 μg/mL BSA; 100 μg/mL tRNA;0.01% Igepal Ca-630; 5% glycerol) or in an identical buffer that lackedbackground tRNA “RNAlessPB”. FIG. 25 shows the activity of Cas13a (SEQID NO: 104) in the DETECTR assay, as measured by fluorescence, for eachof the tested reporters. The “U5” reporter (/5-6FAM/rUrUrUrUrU/3IABkFQ/(SEQ ID NO: 111)) and the “UU” reporter (/56-FAM/TArUrUGC/3IABkFQ/)exhibited the best performance. A reporter with the same nucleotidesequence as the “U5” reporter but with a different fluorophore andquencher, “TYE665U5” (/5-TYE665/rUrUrUrUrU/3IABkRQ/ (SEQ ID NO: 111))also performed well. Increasing the length of the reporter generatedhigher background in processing buffers that did not contain backgroundRNA.

The optimal buffer composition and pH for Cas13a DETECTR assays wasidentified. To determine the ideal buffer and pH for the Cas13adetection assay, 84 different combinations of buffers and pH weretested. The final buffer concentration used in each assay was 20 mM.Aside from the buffer itself, the remaining assay components included 50mM KCl, 5 mM MgCl₂, 10 g/mL BSA, 100 μg/ML tRNA, 0.01% Igepal Ca-630,and 5% Glycerol. Cas13a DETECTR assays were performed with 1 pM targetRNA or no target RNA as a control. The dotted line indicates performanceof a HEPES pH 6.8 Cas13a reaction buffer (also referred to as “HEPES pH6.8 buffer”; HEPES pH 6.8 buffer with tRNA: 20 mM HEPES pH 6.8; 50 mMKCl; 5 mM MgCl₂; 10 μg/mL BSA; 100 μg/mL tRNA; 0.01% Igepal Ca-630; 5%glycerol). Dots indicate replicates. FIG. 26 shows Cas13a activity inthe DETECTR assay, as measured by fluorescence, for each of the testedconditions. These results demonstrated that the optimal pH is around 7.5and that the imidazole, phosphate, tricine, and SPG buffers are all highperforming buffers, in comparison to the HEPES pH 6.8 buffer (20 mMHEPES pH 6.8; 50 mM KCl; 5 mM MgCl₂; 10 μg/mL BSA; 100 μg/mL tRNA; 0.01%Igepal Ca-630 (NP-40); 5% Glycerol). Cas13a detection was inhibited atpH values below 6.5.

Cas13a activity in DETECTR assays was assessed in a variety ofcommercially available buffers. Cas13a detection assays were carried outwith either 1 pM target RNA or no target RNA at 37° C. Reactions wereperformed either in the presence or absence of 100 ng/μL tRNA. Buffersused included NEB1 (NEBuffer1, New England Biolabs (NEB)), NEB2(NEBuffer2, NEB), NEB3 (NEBuffer3, NEB), Cutsmart (NEB), RNPB (RNApolymerase buffer, NEB), and the HEPES pH 6.8 buffer (20 mM HEPES pH6.8; 50 mM KCl; 5 mM MgCl2; 10 μg/mL BSA; 100 μg/mL tRNA; 0.01% IgepalCa-630 (NP-40); 5% Glycerol). These buffer compositions are as follows:NEBuffer 1.1 (1× Buffer Components, 10 mM Bis-Tris-Propane-HCl, 10 mMMgCl₂, 100 μg/ml BSA, pH 7.0@25° C.); NEBuffer 2.1 (1× BufferComponents, 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 100 μg/ml BSA, pH7.9@25° C.); NEBuffer 3.1 (1× Buffer Components, 100 mM NaCl, 50 mMTris-HCl, 10 mM MgCl₂, 100 μg/ml BSA, pH 7.9@25° C.); CutSmart Buffer(1× Buffer Components, 50 mM Potassium Acetate, 20 mM Tris-acetate, 10mM Magnesium Acetate, 100 μg/ml BSA, pH 7.9@25° C.); and 1× RNAPolReaction Buffer (40 mM Tris-HCl, 6 mM MgCl₂, 1 mM DTT, 2 mM spermidine(pH 7.9 @ 25° C.)). The results demonstrated that Cas13a performanceimproved in NEBuffer2 and Cutsmart in comparison to the HEPES pH 6.8buffer (20 mM HEPES pH 6.8; 50 mM KCl; 5 mM MgCl2; 10 μg/mL BSA; 100μg/mL tRNA; 0.01% Igepal Ca-630 (NP-40); 5% Glycerol). FIG. 27 showsCas13a (SEQ ID NO: 104) performance in the DETECTR assay, as measured byfluorescence, for each of the five commercially available buffers and aHEPES pH 6.8 buffer (20 mM HEPES pH 6.8; 50 mM KCl; 5 mM MgCl2; 10 μg/mLBSA; 100 μg/mL tRNA; 0.01% Igepal Ca-630 (NP-40); 5% Glycerol).

Combining the above described observations of buffer performance, anhigh performance Cas13a buffer called MBuffer1 was developed. 1×MBuffer1 include 20 mM imidazole pH 7.5, 50 mM KCl, 5 mM MgCl₂, 10 μg/μLBSA, 0.01% Igepal Ca-630, and 5% glycerol. FIG. 28 shows a comparison ofa HEPES pH 6.8 buffer (“Original Buffer,” 20 mM HEPES pH 6.8; 50 mM KCl;5 mM MgCl2; 10 μg/mL BSA; 100 μg/mL tRNA; 0.01% Igepal Ca-630 (NP-40);5% Glycerol) to the high performance buffer (“MBuffer1,” 20 mM imidazolepH 7.5, 50 mM KCl, 5 mM MgCl₂, 10 μg/μL BSA, 0.01% Igepal Ca-630, and 5%glycerol) for a Cas13a DETECTR assay with serially diluted target RNAsand run at 37° C. for 30 minutes. The limit of detection for the HEPESpH 6.8 buffer was around 1 pM, whereas the limit of detection for thehigh performance buffer was found to be between 100 fM and 10 fM. Thus,FIG. 28 demonstrates that there is a 10× and 100× improvement in assayperformance using the high performance buffer.

Cas13a performance in DETECTR assays was evaluated with and withoutglycerol. Glycerol is commonly used in many enzymatic buffers. Cas13adetection assays with varying concentrations of target RNA were run at37° C. for 30 minutes in either an high performance buffer with glycerol(“MBuffer1,” 20 mM imidazole pH 7.5, 50 mM KCl, 5 mM MgCl₂, 10 μg/μLBSA, 0.01% Igepal Ca-630, and 5% glycerol) or an high performance bufferwithout glycerol (“MBuffer1—no glycerol,” 20 mM imidazole pH 7.5, 50 mMKCl, 5 mM MgCl₂, 10 μg/μL BSA, and 0.01% Igepal Ca-630). FIG. 29 showsthat 5% glycerol in the high performance buffer (“MBuffer1,” left graph)increases performance of a Cas13a (SEQ ID NO: 104) DETECTR assay incomparison to an identical buffer without glycerol (“MBuffer1—noglycerol,” right graph).

Cas13a performance in DETECTR assays was evaluated with varyingconcentrations of BSA and NP-40. BSA and NP-40 (Igecal-Ca 630) are usedin many enzymatic buffers to increase assay performance and decreasebinding of the protein to plastic surfaces. Cas13a DETECTR assays wererun with 1 μM target RNA or no target RNA at 37° C. for 30 minutes in anhigh performance buffer with varying concentrations and combinations ofNP-40 (Igepal Ca-630) and BSA. In addition to the indicatedconcentrations of NP-40 and BSA, each buffer contained 20 mM imidazolepH 7.5, 50 mM KCl, 5 mM MgCl₂, and 5% glycerol. FIG. 30 shows a gradientchart of Cas13a (SEQ ID NO: 104) activity in the DETECTR assay, asmeasured by fluorescence, (darker squares indicate increased Cas13aactivity) versus varying NP-40 concentration along the x-axis andvarying BSA concentration along the y-axis. The results indicated thatboth BSA and NP-40 improve the assay. NP-40 (Igecal-Ca 630) was found tobe important for the efficiency of the Cas13a detection assay. Smallamounts of BSA also improved the performance of the assay.Concentrations of 0.05% to 0.0625% NP-40 were most optimal andconcentrations of 2.5 to 0.625 μg/mL BSA were most optimal. BSA did notimprove assay performance unless NP-40 was also present.

To determine which types of compounds may increase or inhibit theperformance of Cas13a in DETECTR assays, assays were run with 96different additives (JBScreen Plus HTS, Jena Biosciences). Additivesfrom the Jena Biosciences plate were diluted 1:66 into the final Cas13aDETECTR assay with 100 μM of target. FIG. 31 shows Cas13a performance inDETECTR assays, as measured by fluorescence, versus the differentadditives tested. Results showed that the specific compounds thatinhibited the performance of the assay included: beryllium sulfate,manganese chloride, zinc chloride, tri-sodium citrate, copper chloride,yttrium chloride, 1-6-Diaminohexane, 1-8-diaminooctane, ammoniumfluoride, ethanolamine, lithium salicylate, magnesium sulfate, potassiumcyanate, and sodium fluoride.

A buffer developed for LbCas12a (SEQ ID NO: 1) used Tris pH 7.5. FIG. 32shows the results of screening 84 different buffer and pH combinationsto determine the optimal buffer for LbCas12a activity in DETECTR assays,as measured by fluorescence. A final buffer concentration of 20 mM wasused for each assay. The remaining assay components included 100 mM KCl,5 mM MgCl₂, 50 μg/mL heparin, 1 mM DTT, and 5% Glycerol. LbCas12aDETECTR assays were performed at 37° C. with 100 μM target DNA or notarget DNA as a control. The dotted line indicates performance ofLbCas12a in the Tris pH 7.5 buffer (20 mM Tris-HCl, pH 7.5; 100 mM KCl;5 mM MgCl2; 1 mM DTT; 5% glycerol; 50 μg/mL heparin). Dots indicatereplicates. Results of this experiment showed that LbCas12a prefers pH8.0 and works well in AMPD, BIS-TRISpropane, DIPSO, HEPES, MOPS, TAPS,TRIS, and tricine buffers. LbCas12a was inhibited at pH 6.5 and belowand was not functional in phosphate, succinate, malonate, citrate, MES,and ADA buffers.

The optimal salt type and salt concentration was determined for LbCas12aperformance in DETECTR assays. LbCas12a DETECTR assays were run with 10μM of target DNA or no target DNA at 37° C. for 30 minutes with varyingconcentrations of KCl. FIG. 33 shows LbCas12a performance in DETECTRassays, as measured by fluorescence, in each of the tested conditions.Results indicated that the LbCas12a performed best in assays with lowKCl concentrations (0-40 mM or less than 20 mM salt and less KCl). Above80 mM the assay was inhibited, with little to no activity above 160 mM.

The optimal buffer type and pH was determined for the Type V CRISPR-CasCas12 variant (SEQ ID NO: 11) performance in DETECTR assays. FIG. 34shows the performance of SEQ ID NO: 11 in DETECTR assays, as measured byfluorescence, for each of the tested conditions (buffer type and pH).The final concentration of buffer in each assay was 20 mM. The remainingassay components included 120 mM NaCl, 5 mM MgCl₂, and 1% Glycerol. SEQID NO: 11 DETECTR assays were performed at 37° C. with 1 nM target DNAor no target DNA (0 nM) as a control. The dotted line indicates theperformance of Cas12 variant in the Tris pH 7.5 buffer (20 mM Tris-HCl,pH 7.5; 100 mM KCl; 5 mM MgCl2; 1 mM DTT; 5% glycerol; 50 μg/mLheparin). Results showed that SEQ ID NO: 11 performed optimally in a pHof 7.5. High performance buffers included DIPSO, HEPES, MOPS, TAPS,imidazole, and tricine. SEQ ID NO: 11 was inhibited in Tris buffers butwas still functional. SEQ ID NO: 11 showed little or no functionalactivity in succincate, malonate, MES, ADA, citrate, SPG, and phosphatebuffers.

Further investigation of the optimal buffer type and pH was carried outfor SEQ ID NO: 11. Some proteins prefer buffers that have reducednumbers of chloride ions. To determine whether SEQ ID NO: 11 performedbetter in chloride- or acetate-based buffers, a screen of salt type andconcentration was carried out. FIG. 35 shows SEQ ID NO: 11 performancein DETECTR assays, as measured by fluorescence, for the various salttypes and concentrations tested. Assay components included 20 mM HEPESpH 7.3, 1% Glycerol, and 5 mM of MgCl or MgOAc. Varying amounts of KClor KOAc were screened with the corresponding magnesium type. SEQ ID NO:11 detection assays were carried out at 37° C. with 1 nM target DNA and0 nM target DNA as a control for 30 minutes. SEQ ID NO: 11 performedbest at low salt concentrations of around 4 mM (ranging from 2-10 nM)and showed increased activity in buffers with MgOAc and KOAc (acetatebuffers), in comparison to buffers with MgCl and KCl.

The optimal concentrations of heparin and salt concentrations weredetermined for SEQ ID NO: 11, since a relationship was observed betweensalt and heparin for SNP sensitivity using LbCas12a (SEQ ID NO: 1). Thebase buffer included 20 mM HEPES pH 7.3, 5 mM MgOAc, and 1% Glycerol.Varying amounts of KOAc and heparin were screened. SEQ ID NO: 11 DETECTRassays were performed at 37° C. with 1 nM target DNA or no target DNA asa control for 30 minutes. For LbCas12a heparin and salt concentrationscombined to affect the specificity of the enzyme. FIG. 36 shows SEQ IDNO: 11 performance in DETECTR assays, as measured by fluorescence(darker squares indicate greater fluorescence and more activity), versusheparin concentration on the x-axis and KOAc buffer concentration on they-axis. The results of this experiment indicated that SEQ ID NO: 11trans-cleavage activity was inhibited by heparin and SEQ ID NO: 11prefers low salt.

Inhibitors and enhancers of assay performance was evaluated for SEQ IDNO: 11 DETECTR assays. DETECTR assays were run with 96 differentadditives (JBScreen Plus HTS, Jena Biosciences). Additives from the JenaBiosciences plate were diluted 1:66 into a final SEQ ID NO: 11 detectionassays with 1 nM of target. FIG. 37 shows that specific compoundsinhibited the performance of the Cas12 variant (SEQ ID NO: 11) DETECTRassay including: benzamidine hydrochloride, beryllium sulfate, manganesechloride, potassium bromide, sodium iodine, zinc chloride, di-ammoniumhydrogen phosphate, tri-lithium citrate, tri-sodium citrate, cadmiumchloride, copper chloride, yttrium chloride, 1-6 diaminohexane,1-8-diaminooctane, ammonium fluoride, and ammonium sulfate. Compoundsthat increased assay performance included: polyvinyl alcohol type II,DTT, DMSO, polyvinylpyrrolidone K15, polyethylene glycol (PEG) 600, andpolypropylene glycol 400. Concentrations in the legend are listed as thestock concentration. Buffer concentrations in the assay are 2% of theconcentration listed in the figure legend. In addition to the buffersindicated on the x-axis, the remaining assay components included 120 mMNaCl, 5 mM MgCl₂, and 1% glycerol. The dotted line indicates theperformance of the Cas12 variant in the Tris pH 7.5 buffer (20 mMTris-HCl, pH 7.5; 100 mM KCl; 5 mM MgCl2; 1 mM DTT; 5% glycerol; 50μg/mL heparin).

The positions along a target sequence most sensitive to single mutationswas identified by tiling all nucleotide possibility (A, T, C, G) at the20 positions downstream of the PAM motif along a SEQ ID NO: 11 targetsite on HERC2 and ALDH. FIG. 38 shows the results of evaluating SNPsensitivity along target sequences for SEQ ID NO: 11. Purple squaresindicate the WT sequence that matched the crRNA was used to interrogatethe sensitivity of SEQ ID NO: 11 to mutations along a target site onHERC2 and ALDH. Results indicated stronger SNP differentiation for SEQID NO: 11 along the 3′ end of the crRNA (distal from the PAM). A similarcomplementary experiment using LbCas12a using the same sets of targetsites and crRNAs was carried out. FIG. 39 shows the results ofevaluating SNP sensitivity along target sequences for LbCas12a. LbCas12adisplayed strong mutation sensitivity at all positions along HERC2, andsensitivity on the PAM proximal (complementary to the 5′ end of thecrRNA target sequence) on ALDH2. This suggested that LbCas12a was moresensitive to mutations in this region and that mutation sensitivity astarget site dependent.

Example 9 Volumes of Sample and the Detection Reaction

This example describes volumes of sample and the detection reaction ofDETECTR assays provided herein. A first volume containing a sample isprovided. The first volume is contacted to a second volume. The secondvolume contains a guide nucleic acid, a programmable nuclease (e.g., aCas12 or a Cas13), and a reporter. The first volume contains a samplethat is unlysed, a sample that has been lysed, or a sample that has beenlysed and undergone: reverse transcription, amplification, in vitrotranscription, or any combination thereof. The sample contains a bufferfor cell lysis, a buffer for amplification, a primer, a polymerase,target nucleic acid, a non-target nucleic acid, a single-stranded DNA, adouble-stranded DNA, a salt, a buffering agent, an NTP, a dNTP, or anycombination thereof. The first volume is 1 to 5 μL. The second volume is18 to 22 μL. The programmable nuclease is able to efficiently andrapidly cleave a nucleic acid of the reporter and the detectable signalproduced in the presence of a target nucleic acid sequence in the firstvolume is not dampened.

Example 10 Primer Design for Combined LAMP and DETECTR Reactions

This example describes primer design for combined LAMP and DETECTRreactions for amplification and detection of a target nucleic acid, asprovided herein. Strategies for designing primers for use in combinedLAMP and DETECTR reactions were tested and evaluated for multiple targetnucleic acids. From these experiments, a set of design guidelines wasdetermined to facilitate combined LAMP and DETECTR reactions for DNAnucleic acid targets or RT-LAMP and DETECTR reactions for RNA nucleicacid targets.

FIG. 40 shows a scheme for designing primers for loop mediatedisothermal amplification (LAMP) of a target nucleic acid sequence. LAMPgenerates concatemer amplicons, comprising the target nucleic acidsequence, that form from nucleic acid loops during amplification. Togenerate the loops, LAMP may use from four to six primers, including theforward outer primer, the backward outer primer, the forward innerprimer, the backward inner primer, optionally a loop forward primer, andoptionally a loop backward primer.

FIG. 41 shows schematics of exemplary configurations of various regionsof the nucleic acid sequence that correspond to or anneal LAMP primers,guide RNA sequences, protospacer-adjacent motif (PAM) or protospacerflanking site (PFS), and target nucleic acid sequences for amplificationand detection by LAMP and DETECTR.

FIG. 41A shows a schematic of an exemplary arrangement of the guide RNA(gRNA) with respect to the various regions of nucleic acid sequence thatcorrespond to or anneal LAMP primers. In this arrangement, the guide RNAis reverse complementary to a sequence of the target nucleic acid, whichis between an F1c region and a B1 region.

FIG. 41B shows a schematic of an exemplary arrangement of the guide RNAsequence with respect to the various regions of the nucleic acidsequence that correspond to or anneal LAMP primers. In this arrangement,the guide RNA is partially reverse complementary to a sequence of thetarget nucleic acid, which is between an F1c region and a B1 region. Forexample, the target nucleic acid comprises a sequence between an F1cregion and a B1 region that is reverse complementary to at least 60% ofa guide nucleic acid. In this arrangement, the guide RNA is not reversecomplementary to the forward inner primer or the backward inner primershown in FIG. 40.

FIG. 41C shows a schematic of an exemplary arrangement of the guide RNAwith respect to the various regions of the nucleic acid sequence thatcorrespond to or anneal LAMP primers. In this arrangement, the guide RNAhybridizes to a sequence of the target nucleic acid, which is within theloop region between the B1 region and the B2 region. The primersequences do not contain and are not reverse complementary to the PAM orPFS.

FIG. 41D shows a schematic of an exemplary arrangement of the guide RNAwith respect to the various regions of the nucleic acid sequence thatcorrespond to or anneal LAMP primers. In this arrangement, the guide RNAhybridizes to a sequence of the target nucleic acid, which is within theloop region between the F2c region and F1c region. The primer sequencesdo not contain and are not reverse complementary to the PAM or PFS.

Primer sets and guide RNAs for combined LAMP and DETECTR reactions weretested for their sensitivity and specificity to detect the presence of atarget nucleic acid in a sample. DETECTR signal, measured as rawfluorescence, was measured for each LAMP primer set with each of threeguide RNAs designed for the specific LAMP primer set. DETECTR signal wasmeasured in a sample containing 10000 copies of a target nucleic acidsequence and a sample containing zero copies of a target nucleic acidsequence (negative control) for each LAMP primer and guide RNA pair.

FIG. 42 shows schematics of exemplary configurations of various regionsof the nucleic acid sequence that correspond to or anneal LAMP primers,guide RNA sequences, protospacer-adjacent motif (PAM) or protospacerflanking site (PFS), and target nucleic acid sequences for combined LAMPand DETECTR for amplification and detection, respectively. At the right,the schematics also show corresponding fluorescence data using guide RNAsequences to detect the presence of a target nucleic acid sequencefollowing amplification of the target nucleic acid using the LAMPamplification, where a fluorescence signal is the output of the DETECTRreaction and indicates presence of the target nucleic acid. Sequencesand arrangements of the regions that correspond to or anneal LAMPprimers, guide RNA sequences, protospacer-adjacent motif (PAM) orprotospacer flanking site (PFS), and target nucleic acid sequences areillustrated in FIG. 43A-FIG. 43C. Three exemplary guide RNAs (gRNA1 (SEQID NO: 261), gRNA2 (SEQ ID NO: 262), and gRNA3 (SEQ ID NO: 263)) weretested in each primer configuration. Fluorescence signal from theDETECTR reactions, indicative of detection of a target nucleic acid,measured for each of the three guide RNAs was compared for two samples,one containing the target nucleic acid sequence (1000 genome copies perreaction) and a negative control (0 genome copies per reaction) thatdoes not contain the target nucleic acid sequence. Sequences of thegRNAs and the primers are shown below in TABLE 9.

TABLE 9 Exemplary LAMP Primer and DETECTR gRNA Sets SEQ ID NO: NameSequence SEQ ID NO: 201 IAVE-MP-set5-F3 GCGAAAGCAGGTAGATATTGASEQ ID NO: 249 IAVE-MP-set5-F2 ATGAGTCTTCTAACCGAGGT SEQ ID NO: 205IAVE-MP-set5-LF TGACGGGACGATAGAGAGAA SEQ ID NO: 250 IAVE-MP-set5-F1cTTCAAGTCTCTGCGCGATCTC SEQ ID NO: 251 IAVE-MP-set5-B1cTTGAGGCTCTCATGGAATGGC SEQ ID NO: 206 IAVE-MP-set5-LBACAAGACCAATCCTGTCACC SEQ ID NO: 252 IAVE-MP-set5-B2 AGCGTGAACACAAATCCTAASEQ ID NO: 202 IAVE-MP-set5-B3 CATTCCCATTGAGGGCATT SEQ ID NO: 210IAVE-MP-set8-F3 TCTTCTAACCGAGGTCGAA SEQ ID NO: 253 IAVE-MP-set8-F2GAAGATGTCTTTGCAGGGAA SEQ ID NO: 214 IAVE-MP-set8-LFATTCCATGAGAGCCTCAAGATC SEQ ID NO: 254 IAVE-MP-set8-F1cTCAGAGGTGACAGGATTGGTCT SEQ ID NO: 255 IAVE-MP-set8-B1cTTGTGTTCACGCTCACCGTG SEQ ID NO: 215 IAVE-MP-set8-LB GAGGACTGCAGCGTAGACSEQ ID NO: 202 IAVE-MP-set8-B2 CATTCCCATTGAGGGCATT SEQ ID NO: 211IAVE-MP-set8-B3 CTGCTCTGTCCATGTTGTT SEQ ID NO: 256 IAVE-MP-set1-F3GACTTGAAGATGTCTTTGCA SEQ ID NO: 257 IAVE-MP-set1-F2 CAGATCTTGAGGCTCTCSEQ ID NO: 258 IAVE-MP-set1-LF GTCTTGTCTTGTCTTTAGCCA SEQ ID NO: 259IAVE-MP-set1-F1c TTAGTCAGAGGTGACAGGATTG SEQ ID NO: 255 IAVE-MP-set1-BlcTTGTGTTCACGCTCACCGTG SEQ ID NO: 188 IAVE-MP-set1-LB CAGTGAGCGAGGACTGSEQ ID NO: 260 IAVE-MP-set1-B2 TTTGGACAAAGCGTCTACG SEQ ID NO: 184IAVE-MP-set1-B3 TGTTGTTTGGGTCCCCATT SEQ ID NO: 261 gRNA1UUUGUGUUCACGCUCACCGUGCCC SEQ ID NO: 262 gRNA2 UUUAGCCAUUCCAUGAGAGCCUCASEQ ID NO: 263 gRNA3 UUUGGACAAAGCGUCUACGCUGCA

FIG. 42A shows a schematic of an arrangement of various regions of thenucleic acid sequence that correspond to or anneal LAMP primers (SEQ IDNO: 201, SEQ ID NO: 202, SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO:249-SEQ ID NO: 252) and positions of three guide RNAs (gRNA1 (SEQ ID NO:261), gRNA2 (SEQ ID NO: 262), and gRNA3 (SEQ ID NO: 263)) relative tothe LAMP primers (at left). gRNA1 partially overlaps with the B2c regionand is, thus, reverse complementary to a portion of to the B2 region.gRNA2 overlaps with the B1 region and is, thus, reverse complementary tothe B1c region. gRNA3 partially overlaps with the B3 region andpartially overlaps with the B2 region and is, thus, partially reversecomplementary to the B3c region and partially reverse complementary tothe B2c region. The complementary regions (B1, B2c, B3c, F1, F2c, andF3c) are not depicted, but correspond to the regions shown in FIG. 40.At right is a graph of fluorescence from the DETECTR reaction in thepresence of 10,000 genome copies (before amplification) of the targetnucleic acid or 0 genome copies of the target nucleic acid. DETECTRreactions with gRNA1 and gRNA3 exhibited low fluorescence intensity,indicating low to no detection of the target nucleic acid (right). gRNA2produced a fluorescent signal independent of the presence of the targetnucleic acid due to hybridization of gRNA2 with the B1c region of theBIP and self-activation of the guide RNA and Cas cleavage activity.Hybridization of gRNA2 with the BIP may further lead to amplification ofa non-target sequence due to the formation of a primer dimer. Thesequences and arrangements of the regions that correspond to or annealLAMP primers, guide RNA sequences, protospacer-adjacent motif (PAM) orprotospacer flanking site (PFS), and target nucleic acid sequences areshown in FIG. 43A.

FIG. 42B shows a schematic of an arrangement of various regions of thenucleic acid sequence that correspond to or anneal LAMP primers (SEQ IDNO: 202, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 214, SEQ ID NO: 215,SEQ ID NO: 253-SEQ ID NO: 255) and positions of three guide RNAs (gRNA1(SEQ ID NO: 261), gRNA2 (SEQ ID NO: 262), and gRNA3 (SEQ ID NO: 263))relative to the LAMP primers (at left). gRNA1 overlaps with the B1cregion and is, thus, reverse complementary to the B1 region. gRNA2overlaps with the LF region and is, thus, reverse complementary to theLFc region. gRNA 3 partially overlaps with the B2 region and partiallyoverlaps with the LBc region and is, thus, partially reversecomplementary to the B2c region and is partially reverse complementaryto the LB region. At right is a graph of fluorescence from the DETECTRreaction in the presence of 10,000 genome copies (before amplification)of the target nucleic acid or 0 genome copies of the target nucleicacid. All three guide RNAs detected the presence of the target nucleicacid in DETECTR reactions, as evidenced by a high fluorescence signal inthe presence of the target nucleic acid (right). gRNA1 also produced anon-specific fluorescent signal in the absence of the target nucleicacid due to primer-dimer formation with the BIP. gRNA2 and gRNA3 did notproduce a substantial non-specific fluorescent signal. The sequences andarrangements of the regions that correspond to or anneal LAMP primers,guide RNA sequences, protospacer-adjacent motif (PAM) or protospacerflanking site (PFS), and target nucleic acid sequences are shown in FIG.43B.

FIG. 42C shows a schematic of an arrangement of various regions of thenucleic acid sequence that correspond to or anneal LAMP primers (SEQ IDNO: 184, SEQ ID NO: 188, SEQ ID NO: 255-SEQ ID NO: 260) and positions ofthree guide RNAs (gRNA1 (SEQ ID NO: 261), gRNA2 (SEQ ID NO: 262), andgRNA3 (SEQ ID NO: 263)) relative to the LAMP primers (at left). gRNA1overlaps with the B1c region and is, thus, reverse complementary to theB1 region. gRNA2 partially overlaps with the LF region and partiallyoverlaps with the F2c region and is, thus, partially reversecomplementary to the LFc region and partially reverse complementary tothe F2 region. gRNA3 overlaps with the B2 and is, thus, reversecomplementary to the B2c region. At right is a graph of fluorescencefrom the DETECTR reaction in the presence of 10,000 genome copies(before amplification) of the target nucleic acid or 0 genome copies ofthe target nucleic acid. gRNA2 and gRNA3 specifically detected thepresence of the target nucleic acid in DETECTR reactions, as evidencedby a high fluorescence signal in the presence of the target nucleic acidand low fluorescence signal in the absence of the target nucleic acid(right). gRNA1 detected the presence of the target nucleic acid in aDETECTR reaction but also non-specifically produced a fluorescencesignal in the absence of the target nucleic acid due to primer-dimerformation with the BIP, as evidenced by a high fluorescence signal inthe presence of the target nucleic acid and a moderate fluorescencesignal in the absence of the target nucleic acid. The sequences andarrangements of the regions that correspond to or anneal LAMP primers,guide RNA sequences, protospacer-adjacent motif (PAM) or protospacerflanking site (PFS), and target nucleic acid sequences are shown in FIG.43C.

Example 11 Detection of a Target Nucleic Acid with Combined LAMP andDETECTR Reactions

This example describes detection of a target nucleic acid with combinedLAMP and DETECTR reactions. Ten LAMP primer sets (#1-#10) for use inRT-LAMP assays were tested for sensitivity and specificity for samplescontaining a target nucleic acid sequence. Detection following RT-LAMPamplification was performed using either SYTO 9 detection or DETECTR.The sequences of the LAMP primers in each primer set are provided inTABLE 10.

TABLE 10 LAMP Primers for RT-LAMP Amplification and Detection SEQ ID NO:Primer Name Primer Set Sequence SEQ ID NO: 138 F3 RSV-A- #1TGGAACAAGTTGTGGAGG set13 SEQ ID NO: 139 B3 RSV-A- #1TGCAGCATCATATAGATCTTGA set13 SEQ ID NO: 140 FIP RSV-A- #1TAGTGATGCTTTTGGGTTGTTCAAT set13 TGTATGAGTATGCTCAAAAATTGG SEQ ID NO: 141BIP RSV-A- #1 GTGTAGTATTGGGCAATGCTGCTCC set13 TTGGTGTACCTCTGTSEQ ID NO: 142 LF RSV-A- #1 TATGGTAGAATCCTGCTTCTCC set13 SEQ ID NO: 143LB RSV-A- #1 TGGCCTAGGCATAATGGGAGA set13 SEQ ID NO: 144 F3 RSV-A- #2AACAAGTTGTGGAGGTGTA set14 SEQ ID NO: 145 B3 RSV-A- #2CCATTTTCTTTGAGTTGTTCAG set14 SEQ ID NO: 146 FIP RSV-A- #2TAGTGATGCTTTTGGGTTGTTCAAG set14 AGTATGCTCAAAAATTGGGTG SEQ ID NO: 147BIP RSV-A- #2 GTATTGGGCAATGCTGCTGGCATAT set14 AGATCTTGATTCCTTGGTGSEQ ID NO: 148 LF RSV-A- #2 ATATGGTAGAATCCTGCTTCTC set14 SEQ ID NO: 149LB RSV-A- #2 CCTAGGCATAATGGGAGAATAC set14 SEQ ID NO: 144 F3 RSV-A- #3AACAAGTTGTGGAGGTGTA set15 SEQ ID NO: 145 B3 RSV-A- #3CCATTTTCTTTGAGTTGTTCAG set15 SEQ ID NO: 150 FIP RSV-A- #3ATAGTGATGCTTTTGGGTTGTTCAA set15 GTATGCTCAAAAATTGGGTG SEQ ID NO: 151BIP RSV-A- #3 GCTGCTGGCCTAGGCATAATGCATC set15 ATATAGATCTTGATTCCTTSEQ ID NO: 406 LF RSV-A- #3 TATATGGTAGAATCCTGCTTCTC set15 SEQ ID NO: 152LB RSV-A- #3 GGGAGAATACAGAGGTACAC set15 SEQ ID NO: 153 F3 RSV-A- #4GGGTCTTAGCAAAATCAGTT set16 SEQ ID NO: 139 B3 RSV-A- #4TGCAGCATCATATAGATCTTGA set16 SEQ ID NO: 154 FIP RSV-A- #4GAATCCTGCTTCTCCACCCAATTGA set16 CACGCTAGTGTACAAGC SEQ ID NO: 141BIP RSV-A- #4 GTGTAGTATTGGGCAATGCTGCTCC set16 TTGGTGTACCTCTGTSEQ ID NO: 155 LF RSV-A- #4 CCTCCACAACTTGTTCCATTTCT set16 SEQ ID NO: 156LB RSV-A- #4 TGGCCTAGGCATAATGGGAG set16 SEQ ID NO: 157 F3 RSV-A- #5AAGCAGAAATGGAACAAGTT set17 SEQ ID NO: 145 B3 RSV-A- #5CCATTTTCTTTGAGTTGTTCAG set17 SEQ ID NO: 158 FIP RSV-A- #5TAGTGATGCTTTTGGGTTGTTCAGT set17 GGAGGTGTATGAGTATGC SEQ ID NO: 159BIP RSV-A- #5 GTAGTATTGGGCAATGCTGCTGATA set17 TAGATCTTGATTCCTTGGTGSEQ ID NO: 160 LF RSV-A- #5 TGCTTCTCCACCCAATTTTTGA set17 SEQ ID NO: 161LB RSV-A- #5 GCCTAGGCATAATGGGAGAATAC set17 SEQ ID NO: 153 F3 RSV-A- #6GGGTCTTAGCAAAATCAGTT set18 SEQ ID NO: 139 B3 RSV-A- #6TGCAGCATCATATAGATCTTGA set18 SEQ ID NO: 162 FIP RSV-A- #6GAATCCTGCTTCTCCACCCAGACAC set18 GCTAGTGTACAAGC SEQ ID NO: 141 BIP RSV-A-#6 GTGTAGTATTGGGCAATGCTGCTCC set18 TTGGTGTACCTCTGT SEQ ID NO: 155LF RSV-A- #6 CCTCCACAACTTGTTCCATTTCT set18 SEQ ID NO: 156 LB RSV-A- #6TGGCCTAGGCATAATGGGAG set18 SEQ ID NO: 163 F3 RSV-A- #7TACACAGCTGCTGTTCAA set19 SEQ ID NO: 164 B3 RSV-A- #7 GGTAAATTTGCTGGGCATTset19 SEQ ID NO: 165 FIP RSV-A- #7 TTGGAACATGGGCACCCATAAATG set19TCCTAGAAAAAGACGATG SEQ ID NO: 166 BIP RSV-A- #7 CTAGTGAAACAAATATCCACACCCset19 AGCACTGCACTTCTTGAGTT SEQ ID NO: 167 LF RSV-A- #7TTGTAAGTGATGCAGGAT set19 SEQ ID NO: 168 LB RSV-A- #7AGGGACCCTCATTAAGAGTCATG set19 SEQ ID NO: 169 F3 RSV-A- #8ATACACAGCTGCTGTTCA set20 SEQ ID NO: 164 B3 RSV-A- #8 GGTAAATTTGCTGGGCATTset20 SEQ ID NO: 170 FIP RSV-A- #8 TCTGCTGGCATGGATGATTGAATGT set20CCTAGAAAAAGACGATG SEQ ID NO: 166 BIP RSV-A- #8 CTAGTGAAACAAATATCCACACCCset20 AGCACTGCACTTCTTGAGTT SEQ ID NO: 171 LF RSV-A- #8CCCATATTGTAAGTGATGCAGGAT set20 SEQ ID NO: 172 LB RSV-A- #8AGGGACCCTCATTAAGAGTCAT set20 SEQ ID NO: 169 F3 RSV-A- #9ATACACAGCTGCTGTTCA set21 SEQ ID NO: 173 B3 RSV-A- #9 TGGTAAATTTGCTGGGCATset21 SEQ ID NO: 170 FIP RSV-A- #9 TCTGCTGGCATGGATGATTGAATGT set21CCTAGAAAAAGACGATG SEQ ID NO: 174 BIP RSV-A- #9 TGAAACAAATATCCACACCCAAGGset21 GCACTGCACTTCTTGAGTT SEQ ID NO: 175 LF RSV-A- #9CCATATTGTAAGTGATGCAGGAT set21 SEQ ID NO: 176 LB RSV-A- #9GACCCTCATTAAGAGTCATGAT set21 SEQ ID NO: 177 F3 RSV-A- #10 AACATACGTGAACAAACTTCA set22 SEQ ID NO: 178 B3 RSV-A- #10 GCACATATGGTAAATTTGCTGG set22 SEQ ID NO: 179 FIP RSV-A- #10 ACCCATATTGTAAGTGATGCAGGAT set22 AGGGCTCCACATACACAG SEQ ID NO: 180BIP RSV-A- #10  CTAGTGAAACAAATATCCACACCC set22 AAGCACTGCACTTCTTGAGSEQ ID NO: 181 LF RSV-A- #10  TTTCTAGGACATTGTATTGAACAGC set22SEQ ID NO: 182 LB RSV-A- #10  GGGACCCTCATTAAGAGTCATG set22

FIG. 44 shows the times to result of a reverse-transcription LAMP(RT-LAMP) reaction detected using a DNA binding dye. Amplification wasperformed using primer sets #1-#10. Sequences of the primer sets areprovided in TABLE 10 LAMP amplification, measured by an increase in SYTO9 fluorescence, was observed over time, and time to result wasdetermined as the time to reach half maximum SYTO 9 fluorescenceintensity. Time to result was compared for ten LAMP primer sets in thepresence (1000 genome copies) or absence (0 genome copies) of a targetsequence from an RNA virus. Primer sets, namely #1 (SEQ ID NO: 138-SEQID NO: 143), #7 (SEQ ID NO: 163-SEQ ID NO: 168), #8 (SEQ ID NO: 164, SEQID NO: 166, and SEQ ID NO: 169-SEQ ID NO: 172), and #10 (SEQ ID NO:177-SEQ ID NO: 182), showed clear differentiation between a samplecontaining the target sequence and a negative control lacking the targetsequence. A decreased time to result is indicative of a sample positivefor the target nucleic acid sequence.

FIG. 45 shows fluorescence signal from a DETECTR reaction using a Cas12variant (SEQ ID NO: 11) following a five-minute incubation with productsfrom RT-LAMP reactions. Amplification was performed using primer sets#1-#10. LAMP primer sets #1-6 were designed for use with guide RNA #2(SEQ ID NO: 240), and LAMP primer sets #7-10 were designed for use withguide RNA #1 (SEQ ID NO: 239). Sequences of primers in each primer setare provided in TABLE 10. DETECTR signal was compared for each LAMPprimer set in the presence (1000 genome copies) or absence (0 genomecopies) of a target sequence using either a guide RNA having a sequencecorresponding to SEQ ID NO: 239 (guide RNA #1, top bar graph) or guideRNA having a sequence corresponding to SEQ ID NO: 240 (guide RNA #2,bottom bar graph). Data shows clean differentiation between reactionswith the target sequence and no target control reactions when usingDETECTR to differentiate between specific and non-specific LAMPamplification. The sequences of the gRNAs used in the DETECTR reactionare provided in TABLE 11.

TABLE 11 DETECTR gRNAs for RT-LAMP Amplification with DETECTR SEQ ID NO:gRNA Name Sequence SEQ ID NO: 239 gRNA #1 UAAUUUCUACUAAGUGUAGAUC (R1118)UUAUAAAAGAACUAGCCAA SEQ ID NO: 240 gRNA #2 UAAUUUCUACUAAGUGUAGAUA (R288)CUCAAUUUCCUCACUUCUC

Example 12 Amplifying Influenza A and B Virus Using RT-LAMP and SYTO 9

This example describes amplifying influenza A and B virus using LAMP andSYTO 9. Samples containing either 0, 100, 1000, 10,000, or 100,000copies of an influenza A virus (IAV) or 0, 100, 1000, 10,000, or 100,000copies of an influenza B virus (IBV) target nucleic acid sequence weresubjected to RT-LAMP amplification using different sets of LAMP primers.Sets of LAMP primers (1, 2, 4, 5, 6, 7, 8, 9, 10, 11, or a negativecontrol) were compared for their ability to specifically amplify thetarget nucleic acid sequence. Amplification was measured as a time toresult using SYTO 9. A decreased time to result is indicative of asample positive for the target nucleic acid sequence.

Each reaction RT-LAMP reaction was performed in the presence of 1×NEBIsoAmp Buffer, 4.5 mM MgSO₄, 6.4 U/μL Bst 2.0 (NEB), 0.75 μL WarmstartRTx reverse transcriptase, 1 μL 10× primer mix, and 0.2 μL SYTO 9 per 10μL reaction in nuclease free water.

FIG. 46 shows detection of sequences from influenza A virus (IAV) usingSYTO 9 (a DNA binding dye) following RT-LAMP amplification with LAMPprimer sets 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, or a negative control. Tenreactions were performed per primer set and reactions were performed induplicate. Individual plots depict fluorescence intensity over timeduring the LAMP amplification reaction. Fluorescence from SYTO 9 wasmeasured over time as a function of an amount of target sequence presentin the reaction. Plots in rows show amplification in the presence of,from top to bottom, 0, 100, 1000, 10,000, or 100,000 copies of thetarget nucleic acid. Plots in columns show amplification using, fromleft to right, primer sets 1, 2, IBV, 4, 5, 6, 7, 8, 9, 10, and 11.Primer set 1 (SEQ ID NO: 183-SEQ ID NO: 188) shows a flat negativecontrol curve, indicating suitability for use in LAMP amplificationreactions. Primer set 2 (SEQ ID NO: 189-SEQ ID NO: 194) is well-suitedfor use in amplifying a target nucleic acid using LAMP. Primer set 8(SEQ ID NO: 210-SEQ ID NO: 215) and primer set 10 (SEQ ID NO: 211, SEQID NO: 213-SEQ ID NO: 215, and SEQ ID NO: 219-SEQ ID NO: 220) also workwell in amplifying a target nucleic acid using LAMP. Primer set 8produces a lower negative control amplification signal than primer set10. FIG. 48 shows the time to amplification of an IAV target sequencefollowing LAMP amplification with different primer sets as determinedfrom the SYTO 9 fluorescence traces shown in FIG. 46. Time to result wasdetermined as the time to reach half maximum SYTO 9 fluorescenceintensity. Amplification was detected using SYTO 9 in the presence ofincreasing concentrations of the target nucleic acid sequence (0, 100,1000, 10,000, or 100,000 genome copies of the target sequence perreaction). The assay was capable of distinguishing between negativecontrol reactions (no target sequence) and reactions containing 100,000genome copies of the target sequence for all primer sets. The sequencesof the LAMP primers in each primer set are provided in TABLE 12.

TABLE 12 Primers for Amplification and Detection ofIAV and IBV Virus using RT-LAMP SEQ ID NO: Primer Name Primer SetSequence SEQ ID NO: 183 IAV-MP-F3 #1 GACTTGAAGATGTCTTTGC SEQ ID NO: 184IAV-MP B3 #1 TGTTGTTTGGGTCCCCATT SEQ ID NO: 185 IAV-MP-FIP #1TTAGTCAGAGGTGACAGGATTGCA GATCTTGAGGCTCTC SEQ ID NO: 186 IAV-MP-BIP #1TTGTGTTCACGCTCACCGTGTTTGG ACAAAGCGTCTACG SEQ ID NO: 187 IAV-MP FL #1GTCTTGTCTTTAGCCA SEQ ID NO: 188 IAV-MP BL #1 CAGTGAGCGAGGACTGSEQ ID NO: 189 IAV F3 v2 #2 ACCGAGGTCGAAACGT SEQ ID NO: 190 IAV B3 v2 #2GGTCCCCATTCCCATTG SEQ ID NO: 191 IAV FIP v2 #2 CAAAGACATCTTCAAGTCTCTGCGTTTTTTCTCTCTATCGTCCCGTCA SEQ ID NO: 192 IAV BIP v2 #2AATGGCTAAAGACAAGACCAATCC TTTTTTGTCTACGCTGCAGTCC SEQ ID NO: 193 IAV LF v2#2 CGATCTCGGCTTTGAGGG SEQ ID NO: 194 IAV LB v2 #2 TCACCGTGCCCAGTGAGSEQ ID NO: 195 IAV F3 v3 #3 CGAAAGCAGGTAGATATTGAAAG SEQ ID NO: 196IAV B3 v3 #3 TCTACGCTGCAGTCCTC SEQ ID NO: 197 IAV FIP v3 #3TCAAGTCTCTGCGCGATCTCTTTTT TGAGTCTTCTAACCGAGGT SEQ ID NO: 198 IAV BIP v3#3 AGATGTCTTTGCAGGGAAAAACAC TTTTTTCACAAATCCTAAAATCCCC TTAGSEQ ID NO: 199 IAV LF v3 #3 GACGATAGAGAGAACGTACGTTTC SEQ ID NO: 200IAV LB v3 #3 AAGACCAATCCTGTCACCTCT SEQ ID NO: 201 IAV-set4-F3 #4GCGAAAGCAGGTAGATATTGA SEQ ID NO: 202 IAV-set4-B3 #4 CATTCCCATTGAGGGCATTSEQ ID NO: 203 IAV-set4-FIP #4 CTTCAAGTCTCTGCGCGATCTATGAGTCTTCTAACCGAGGT SEQ ID NO: 204 IAV-set4-BIP #4TTGAGGCTCTCATGGAATGGCAGC GTGAACACAAATCCTAA SEQ ID NO: 205 IAV-set4-LF #4TGACGGGACGATAGAGAGAA SEQ ID NO: 206 IAV-set4-LB #4 ACAAGACCAATCCTGTCACCSEQ ID NO: 201 IAV-set5-F3 #5 GCGAAAGCAGGTAGATATTGA SEQ ID NO: 202IAV-set5-B3 #5 CATTCCCATTGAGGGCATT SEQ ID NO: 207 IAV-set5-FIP #5TTCAAGTCTCTGCGCGATCTCATG AGTCTTCTAACCGAGGT SEQ ID NO: 204 IAV-set5-BIP#5 TTGAGGCTCTCATGGAATGGCAGC GTGAACACAAATCCTAA SEQ ID NO: 205 IAV-set5-LF#5 TGACGGGACGATAGAGAGAA SEQ ID NO: 206 IAV-set5-LB #5ACAAGACCAATCCTGTCACC SEQ ID NO: 201 IAV-set6-F3 #6 GCGAAAGCAGGTAGATATTGASEQ ID NO: 208 IAV-set6-B3 #6 TTGGACAAAGCGTCTACG SEQ ID NO: 203IAV-set6-FIP #6 CTTCAAGTCTCTGCGCGATCTATG AGTCTTCTAACCGAGGTSEQ ID NO: 204 IAV-set6-BIP #6 TTGAGGCTCTCATGGAATGGCAGCGTGAACACAAATCCTAA SEQ ID NO: 205 IAV-set6-LF #6 TGACGGGACGATAGAGAGAASEQ ID NO: 206 IAV-set6-LB #6 ACAAGACCAATCCTGTCACC SEQ ID NO: 201IAV-set7-F3 #7 GCGAAAGCAGGTAGATATTGA SEQ ID NO: 202 IAV-set7-B3 #7CATTCCCATTGAGGGCATT SEQ ID NO: 209 IAV-set7-FIP #7AAGTCTCTGCGCGATCTCGATGAG TCTTCTAACCGAGGT SEQ ID NO: 204 IAV-set7-BIP #7TTGAGGCTCTCATGGAATGGCAGC GTGAACACAAATCCTAA SEQ ID NO: 205 IAV-set7-LF #7TGACGGGACGATAGAGAGAA SEQ ID NO: 206 IAV-set7-LB #7 ACAAGACCAATCCTGTCACCSEQ ID NO: 210 IAV-set8-F3 #8 TCTTCTAACCGAGGTCGAA SEQ ID NO: 211IAV-set8-B3 #8 CTGCTCTGTCCATGTTGTT SEQ ID NO: 212 IAV-set8-FIP #8TCAGAGGTGACAGGATTGGTCTGA AGATGTCTTTGCAGGGAA SEQ ID NO: 213 IAV-set8-BIP#8 TTGTGTTCACGCTCACCGTCATTCC CATTGAGGGCATT SEQ ID NO: 214 IAV-set8-LF #8ATTCCATGAGAGCCTCAAGATC SEQ ID NO: 215 IAV-set8-LB #8 GAGGACTGCAGCGTAGACSEQ ID NO: 216 IAV-set9-F3 #9 TTCTCTCTATCGTCCCGTC SEQ ID NO: 211IAV-set9-B3 #9 CTGCTCTGTCCATGTTGTT SEQ ID NO: 217 IAV-set9-FIP #9CCCTTAGTCAGAGGTGACAGGAAC ACAGATCTTGAGGCTCT SEQ ID NO: 213 IAV-set9-BIP#9 TTGTGTTCACGCTCACCGTCATTCC CATTGAGGGCATT SEQ ID NO: 218 IAV-set9-LF #9GGTCTTGTCTTTAGCCATTCCA SEQ ID NO: 215 IAV-set9-LB #9 GAGGACTGCAGCGTAGACSEQ ID NO: 219 IAV-set10-F3 #10  GTCTTCTAACCGAGGTCGA SEQ ID NO: 211IAV-set10-B3 #10  CTGCTCTGTCCATGTTGTT SEQ ID NO: 220 IAV-set10-FIP #10 GAGGTGACAGGATTGGTCTTGTTG AAGATGTCTTTGCAGGG SEQ ID NO: 213 IAV-set10-BIP#10  TTGTGTTCACGCTCACCGTCATTCC CATTGAGGGCATT SEQ ID NO: 214 IAV-set10-LF#10  ATTCCATGAGAGCCTCAAGATC SEQ ID NO: 215 IAV-C0-LB #10 GAGGACTGCAGCGTAGAC SEQ ID NO: 221 IAV-set11-F3 #11  AAGAAGACAAGAGATATGGCSEQ ID NO: 222 IAV-set11-B3 #11  CAATTCGACACTAATTGATGGC SEQ ID NO: 223IAV-set11-FIP #11  GTCTCCTTGCCCAATTAGCAAGCA TCAATGAACTGAGCASEQ ID NO: 224 IAV-set11-BIP #11  GTGGTGTTGGTAATGAAACGAAGCTGTCTGGCTGTCAGTA SEQ ID NO: 225 IAV-set11-LF #11  ACATTAGCCTTCTCTCCTTTSEQ ID NO: 226 IAV-set11-LB #11  AACGGGACTCTAGCATACT SEQ ID NO: 227M605 F3 IBV IBV AGGGACATGAACAACAAAGA LAMP SEQ ID NO: 228 M606 B3 IBV IBVCAAGTTTAGCAACAAGCCT LAMP SEQ ID NO: 229 M607 FIP IBV IBVTCAGGGACAATACATTACGCATAT LAMP CGATAAAGGAGGAAGTAAACACT CA SEQ ID NO: 230M608 BIP IBV IBV TAAACGGAACATTCCTCAAACACC LAMP ACTCTGGTCATATGCATTCSEQ ID NO: 231 M609 LF IBV IBV TCAAACGGAACTTCCCTTCTTTC LAMPSEQ ID NO: 232 M610 LB IBV IBV GGATACAAGTCCTTATCAACTCTG LAMP C

FIG. 47 shows the time to amplification of an influenza B virus (IBV)target sequence following RT-LAMP amplification. Amplification wasdetected using SYTO 9 in the presence of increasing concentrations oftarget sequence (0, 100, 1000, 10,000, or 100,000 copies of the targetsequence per reaction). RT-LAMP amplification was performed using primerset #8 (SEQ ID NO: 210-SEQ ID NO: 215), provided in TABLE 12.

Example 13 Detection of Influenza a Virus Using LAMP and DETECTR

This example describes detection of influenza A virus using LAMP andDETECTR. Samples containing an influenza A virus (IAV) target nucleicacid sequence or lacking the IAV target nucleic acid sequence weresubjected to RT-LAMP amplification using different sets of LAMP primers.Sets of LAMP primers were compared for their ability to specificallyamplify the target nucleic acid sequence. Presence or absence of thetarget nucleic acid in the sample was subsequently measured usingDETECTR. DETECTR signal, measured by an increase in fluorescent signalupon activation of a programmable nuclease, was observed over time. Anincrease in fluorescence indicates the presence of the target nucleicacid sequence.

Each RT-LAMP reaction was performed in the presence of 1×NEB IsoAmpBuffer, 4.5 mM MgSO₄, 1.4 mM dNTPs (NEB), 6.4 U/μL Bst 2.0 (NEB), 1.5 μLWarmstart RTx, and 2 μL 10× primer mix per 20 μL reaction innuclease-free water. Each DETECTR reaction was performed in the presenceof 1× Processing Buffer, 250 nM crRNA, and 200 nM Sr-WT LbCas12aprogrammable nuclease in nuclease-free water.

FIG. 49 shows detection of target nucleic acid sequences from influenzaA virus (IAV) using DETECTR following RT-LAMP amplification with LAMPprimer sets 1, 2, 4, 5, 6, 7, 8, 9, 10, or a negative control. RT-LAMPamplification was performed using the primer sets provided in TABLE 12.Ten reactions were performed per primer set. DETECTR was performed withdifferent gRNAs. The sequences of the gRNAs used in the DETECTR reactionare provided in TABLE 13. DETECTR signal was measured as a function ofan amount of target sequence present in the reaction. Individual plotsdepict fluorescence intensity over time during DETECTR reactionfollowing LAMP amplification. Individual traces on each plot showamplification followed by DETECTR with a guide RNA corresponding to SEQID NO: 241 (R283 gRNA, blue), a guide RNA corresponding to SEQ ID NO:242 (R781 gRNA, red), a guide RNA corresponding to SEQ ID NO: 243 (R782gRNA, green), or a guide RNA corresponding to SEQ ID NO: 244 (IBV gRNA,purple). Plots in rows show DETECTR following LAMP amplification in thepresence of, from top to bottom, 0, 100, 1000, 10,000, or 100,000 copiesof the target nucleic acid. Plots in columns show DETECTR following LAMPamplification using, from left to right, primer sets 1, 2, 4, 5, 6, 7,8, 9, 10, 11, or IBV. Using primer set 1 resulted in robustamplification of the target nucleic acid by RT-LAMP. Primer set 2 wasalso found to be well-suited for use in combined methods of amplifying atarget nucleic acid sequence by RT-LAMP and detecting the target nucleicacid sequence by DETECTR. Primer set 8 (SEQ ID NO: 210-SEQ ID NO: 215)and primer set 10 (SEQ ID NO: 211, SEQ ID NO: 213-SEQ ID NO: 215, andSEQ ID NO: 219-SEQ ID NO: 220) were well suited for use in combinedRT-LAMP and DETECTR reactions when detected using the guide RNAcorresponding to SEQ ID NO: 243 (gRNA R782), as indicated by robustamplification and detection of the target nucleic acid withoutnon-specific amplification or detection in the absence of the targetnucleic acid. Target nucleic acid sequences from IBV were also detectedby DETECTR after RT-LAMP amplification of the target.

TABLE 13 DETECTR gRNAs for RT-LAMP Amplificationwith DETECTR of IAV or IBV gRNA SEQ ID NO: Name Sequence SEQ ID NO: 241R283 UAAUUUCUACUAAGUGUAGAU UGUUCACGCUCACCGUGCCC SEQ ID NO: 242 R781UAAUUUCUACUAAGUGUAGAU GCCAUUCCAUGAGAGCCUCA SEQ ID NO: 243 R782UAAUUUCUACUAAGUGUAGAU GACAAAGCGUCUACGCUGCA SEQ ID NO: 244 IBVUAAUUUCUACUAAGUGUAGAU (R778) CUAACACUCUCAGGGACAAU

Example 14 Detection of a SNP Using LAMP and DETECTR

This example describes detection of a SNP using LAMP and DETECTR.Strategies for designing primers for use in combined LAMP and DETECTRreactions to detect SNPs were tested and evaluated for multiple targetSNPs. From these experiments, a set of design guidelines was determinedto facilitate combined LAMP and DETECTR reactions for DNA nucleic acidtargets or RT-LAMP and DETECTR reactions for RNA nucleic acid targets.

FIG. 50 shows a scheme for designing primers for LAMP amplification of atarget nucleic acid sequence and detection of a single nucleotidepolymorphism (SNP) in the target nucleic acid sequence. In an exemplaryarrangement, the SNP of the target nucleic acid is positioned betweenthe F1c region and the B1 region.

FIG. 51 shows schematics of exemplary arrangements of LAMP primers,guide RNA sequences, protospacer-adjacent motif (PAM) or protospacerflanking site (PFS), and target nucleic acids with a SNP for methods ofLAMP amplification of a target nucleic acid and detection of the targetnucleic acid using DETECTR.

FIG. 51A shows a schematic of an exemplary arrangement of the guide RNAwith respect to various regions of the nucleic acid sequence thatcorrespond to or anneal LAMP primers. In this arrangement, the PAM orPFS of the target nucleic acid is positioned between an F1c region and aB1 region. The entirety of the guide RNA sequence may be between the F1cregion and the B1c region. The SNP is shown as positioned within asequence of the target nucleic acid that hybridizes to the guide RNA.

FIG. 51B shows a schematic of an exemplary arrangement of the guide RNAsequence with respect to various regions of nucleic acid sequence thatcorrespond to or anneal LAMP primers. In this arrangement, the PAM orPFS of the target nucleic acid is positioned between an F1c region and aB1 region and the target nucleic acid comprises a sequence between anF1c region and a B1 region that is reverse complementary to at least 60%of a guide nucleic acid. In this example, the guide RNA is not reversecomplementary to the forward inner primer or the backward inner primer.The SNP is shown as positioned within a sequence of the target nucleicacid that hybridizes to the guide RNA.

FIG. 51C shows a schematic of an exemplary arrangement of the guide RNAsequence with respect to various regions of the nucleic acid sequencethat correspond to or anneal LAMP primers. In this arrangement, the PAMor PFS of the target nucleic acid is positioned between the F1c regionand the B1 region and the entirety of the guide RNA sequence is betweenthe F1c region and the B1 region. The SNP is shown as positioned withina sequence of the target nucleic acid that hybridizes to the guide RNA.

FIG. 52 shows an exemplary sequence of a nucleic acid comprising two PAMsites and a HERC2 SNP. The positions of two gRNAs targeting the HERC2 ASNP allele at either position 9 with respect to a first PAM site (SEQ IDNO: 245) or at position 14 with respect to a second PAM site (SEQ ID NO:247) are shown. The position of a SNP is indicated with a triangle. TheSNP is positioned at position 9 relative to a first PAM site or position14 relative to a second PAM site. The target sequence is shown in thefigure. The top strand has a sequence of5′-CCAGTTTCATTTGAGCATTAAGTGTCAAGTTCTG-3′ (SEQ ID NO: 750) and the bottomstrand has a sequence of 5′-CAGAACTTGACACTTAATGCTCAAATGAAACTGG-3′ (SEQID NO: 751).

FIG. 53 shows results from DETECTR reactions to detect a HERC2 SNP atposition 9 relative to a first PAM site or position 14 relative to asecond PAM site following LAMP amplification. The SNP position isindicated by a triangle. Fluorescence signal, indicative of detection ofthe target sequence, was measured over time in the presence of a targetsequence comprising either a G SNP allele or an A SNP allele in ITERC2.The target nucleic acid comprising the SNP was amplified using theprimers presented in TABLE 14.

TABLE 14 LAMP Primers for Amplification and Detection of a HERC2 SNPPrimer SEQ ID NO: Primer Name Set Sequence SEQ ID NO: 233 M948 F3 HERC2CTTGTAATCAACATCAGGGTAA HERC2 set3 SEQ ID NO: 234 M949 B3 HERC2AGAAACGACAAGTAGACCATT HERC2 set3 SEQ ID NO: 235 M950 FIP HERC2CGCCTCTTGGATCAGACACATGTG HERC2 set3 TTAATACAAAGGTACAGGA SEQ ID NO: 236M951 BIP HERC2 CACGCTATCATCATCAGGGGCTGC HERC2 set3 TTCAAGTGTATATAAACTCACSEQ ID NO: 237 M952 LF HERC2 GAGAGCCATGAAGAACAAATTCT HERC2 set3SEQ ID NO: 238 M953 LB HERC2 CGAGGCTTCTCTTTGTTTTTAAT HERC2 set3

The target sequence was detected using a guide RNA (crRNA only) todetect either the A allele with the first PAM site (SNP Position 9, “ASNP”), the G allele with the first PAM site (SNP Position 9, “G SNP”),the A allele with the second PAM site (SNP Position 14, “A SNP”) or theG allele with the second PAM site (SNP Position 14, “G SNP”). Four guideRNAs designed for each condition were used. The guide RNAs used for thedetection of the two SNP alleles relative to the two PAM sites arepresented in TABLE 15. The guide RNA corresponding to SEQ ID NO: 245 wasdesigned to detect the A allele at position 9, the guide RNAcorresponding to SEQ ID NO: 246 was designed to detect the G allele atposition 9, the guide RNA corresponding to SEQ ID NO: 247 was designedto detect the A allele at position 14, and the guide RNA correspondingto SEQ ID NO: 248 was designed to detect the G allele at position 14. Ahigh fluorescence signal was detected for the G allele in the presenceof the position 9 G SNP guide RNA (SEQ ID NO: 246, top left) and the Aallele in the presence of the position 9 A SNP guide RNA (SEQ ID NO:245, bottom right). Minimal fluorescence signal was detected for the Gallele in the presence of the position 9 A SNP guide RNA (SEQ ID NO:245, top right) and the position 9 A allele in the presence of the G SNPguide RNA (SEQ ID NO: 246, bottom left). This indicates that theposition 9 G SNP and position 9 A SNP guide RNAs show specificity forthe G allele and A allele, respectively. The position 14 A SNP guide RNA(SEQ ID NO: 247) and the position 14 G SNP guide RNA (SEQ ID NO: 248)detected both alleles, as shown by high fluorescence signal whendetecting the SNP with the position 14 A SNP or G SNP guide RNAs,independent of the target sequence present.

FIG. 54 shows a heatmap of fluorescence from a DETECTR reactionfollowing LAMP amplification of the target nucleic acid sequence. TheDETECTR reaction differentiated between two HERC2 SNP alleles atposition 9 with respect to the PAM, using guide RNAs (crRNA only)specific for the A allele (SEQ ID NO: 245) or the G allele (SEQ ID NO:246). Positive detection is indicated by a high fluorescence value inthe DETECTR reaction. Guide RNA corresponding to SEQ ID NO: 245 wasspecific for A allele, as indicated by (i) a high fluorescence signal inthe A SNP positive control, the HeLa sample, and Sample 2, and (ii) lowfluorescence signal in the G SNP positive control, the negative control,and Sample 1. Guide RNA corresponding to SEQ ID NO: 246 was specific forG allele, as indicated by (i) a high fluorescence signal in the G SNPpositive control, the HeLa sample, and Sample 1, and (ii) lowfluorescence signal in the A SNP positive control, the negative control,and Sample 2. Sample 1 was homozygous for the G allele and Sample 2 washomozygous for the A allele.

TABLE 15 DETECTR Guide RNAs for Amplificationand Detection of a HERC2 SNP SEQ ID NO: gRNA Name SequenceSEQ ID NO: 245 A SNP Position 9 UAAUUUCUACUAAGUGUAGAUAGCAUUAAAU (R570)GUCAAGUUCU SEQ ID NO: 246 G SNP Position 9UAAUUUCUACUAAGUGUAGAUAGCAUUAAGU (R571) GUCAAGUUCU SEQ ID NO: 247A SNP Position 14 UAAUUUCUACUAAGUGUAGAUAUUUGAGCAU (R1138) UAAAUGUCAASEQ ID NO: 248 G SNP Position 14 UAAUUUCUACUAAGUGUAGAUAUUUGAGCAU (R1139)UAAGUGUCAA

FIG. 55 shows combined LAMP amplification of a target nucleic acid byLAMP and detection of the target nucleic acid by DETECTR. Detection wascarried out visually with DETECTR by illuminating the samples with a redLED. Each reaction contained a target nucleic acid sequence comprising aSNP allele for either a blue eye phenotype (“Blue Eye”) or a brown eyephenotype (“Brown Eye”). Samples “Brown*” and “Blue*” were an A allelepositive control and a G allele positive control, respectively. Aposition 9 guide RNA for either the brown eye phenotype (SEQ ID NO: 245,“Br”) or the blue eye phenotype (SEQ ID NO: 246, “B1”) was used for eachLAMP DETECTR reaction. The presence of either the blue eye allele or thebrown eye allele was visually detected by eye, as shown by an increasein fluorescence in each tube containing a target nucleic acid sequenceand a corresponding guide RNA. The guide RNA for the brown eye allelephenotype (SEQ ID NO: 245) was specific for the A allele, as shown by ahigh fluorescence signal (brighter tubes) in tubes containing the browneye guide RNA and either the brown eye target nucleic acid or the A SNPpositive control, and low fluorescence signal (darker tubes) in tubescontaining the brown eye guide RNA and either the blue eye targetnucleic acid or the G SNP positive control. The guide RNA for the blueeye allele (SEQ ID NO: 246) was specific for the G allele, as shown by ahigh fluorescence signal (brighter tubes) in tubes containing the blueeye guide RNA and either the blue eye target nucleic acid or the G SNPpositive control, and low fluorescence signal (darker tubes) in tubescontaining the blue eye guide RNA and either the brown eye targetnucleic acid or the A SNP positive control.

Example 15 High Specificity Buffer

This example shows a high specificity buffer comprising 100 mM NaCl and50 μg/ml heparin enhances the targeting specificity and enhanced SNPdiscrimination capabilities of LbCas12. FIG. 56A-FIG. 56H shows highsensitivity and high specificity buffers for LbCas12a (SEQ ID NO: 1). Inthe presence of 50 μg/ml heparin and 100 mM salt, Cas12a has improvedtargeting specificity and enhanced SNP discrimination capabilities.Target sequences were detected using a crRNA directed to the EGFR wildtype sequence (SEQ ID NO: 448) or a crRNA directed to the EGFR mutantsequence (G SNP, SEQ ID NO: 449). In the absence of heparin and salt,Cas12a has improved sensitivity. For all SNP-related studies, highspecificity buffer was used.

Example 16 Detection of the EGFR SNP T790M (c.2369C>T)

This example shows that Cas12a can be used to detect a single nucleotidepolymorphism (SNP) versus wild-type (WT) of EGFR. The EGFR SNP detectedwas the SNP T790M (c.2369C>T). The sample comprised both the C SNP (WT)and the T SNP (T790M) cell free DNA EGFR DNA standards. Guide RNAsequences for Cas12a detection of the SNPs described in this example arelisted in TABLE 17.

FIG. 57 shows a schematic of PCR primers and guide RNA targetingsequence for EGFR T790M SNP. The forward primer represents a PAM primer(SEQ ID NO: 396), also referred to as a PAMplification primer, whichembeds a PAM sequence (‘TTTV’) upstream of the targeting sequence andincludes a 6 nt 3′ extension for priming. The PAM sequence is requiredfor Cas12a-guide RNA to recognize the matching DNA target. In thisschematic, the guide RNA was designed to target the mismatch located 7nt downstream of the 5′ end of the target sequence (SEQ ID NO: 400).This guide RNA/primer design is used for FIG. 59-FIG. 61.

FIG. 58A-FIG. 58C shows PAMplification F primers (PAM F primers) withvarying 3′ extensions (4 nt in FIG. 58A, 5 nt in FIG. 58B, 6 nt in FIG.58C, SEQ ID NO: 394, SEQ ID NO: 395, and SEQ ID NO: 396, respectively)tested with guide RNA targeting T790M with a mismatch at the 7^(th)position (SEQ ID NO: 400). The PAMplification F primer with 6 ntextension demonstrated optimal detection with the guide RNA. ThisPAMplification F primer was used for FIG. 59-FIG. 63.

FIG. 59A-FIG. 59C illustrates that Cas12 guide RNAs designed to target awild type sequence (“WT” C SNP allele) and sequence comprising a T790M TSNP allele show specific Cas12-based detection in the presence ofcognate single nucleotide polymorphism (SNP). Targets were detected witha crRNA directed to the wild type sequence (SEQ ID NO: 423) or a crRNAdirected to the T SNP allele sequence (SEQ ID NO: 439). Time coursesshow activation of the WT or mutant crRNA only in the presence of thematching target (FIG. 59A and FIG. 59B). A heatmap represents timecourse data at t=60 min (FIG. 59C) n=3 technical replicates; syntheticoligo targets; bars represent mean±SD.

FIG. 60A-FIG. 60D show that Cas12a can detect down to 0.1-1% minorallele frequency (MAF) of EGFR T790M (T SNP allele) in mock cfDNAsamples (Horizon Discovery), with 2 ng of total DNA input and a PCRpre-amplification step. Targets were detected with a crRNA directed tothe wild type sequence (SEQ ID NO: 423) or a crRNA directed to the T SNPallele sequence (SEQ ID NO: 439). Detection of WT (C SNP allele) andmutant allele at t=90 min with low frequency EGFR standards is shown inFIG. 60A. Bar graphs of mutant allele detection only is shown in FIG.60B. A heat map representation of WT and mutant allele detection isshown in FIG. 60C. Samples were run with n=3 replicates and statisticalsignificance was determined by a two-tailed Student's t-test, with*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and bars representing meanplus SD. FIG. 60D shows the different percentages of the WT and mutantallele in sample in a single test tube as pictorial representation ofthe percentage of MAF in the samples tested. The turnaround time was 90minutes and the assay volume was 20 μL.

FIG. 61 shows limit of detection studies illustrating that 2 ng totalDNA is the minimum input allowed for detection of 0.1-1% minor allelefrequency (MAF) of EGFR T790M (T SNP allele) in mock cfDNA samples(Horizon Discovery) with a PCR pre-amplification step. Samples were runwith n=3 replicates and statistical significance was determined by atwo-tailed Student's t-test, with *p<0. 05, **p<0.01, ***p<0.001,****p<0.0001, and bars representing mean plus SD. Targets were detectedusing 7 mm guide RNA directed to T SNP allele (SEQ ID NO: 403). Targetswere amplified using primers corresponding to SEQ ID NO: 396 and SEQ IDNO: 397. FIG. 62 shows a table of FIG. 61 assay parameters.

TABLE 16 DNA sequences used in this study Description Sequence (5′4 3′)EGFR T790M PAMplification F primer (4 nt extension)TCACCTCCACCGTGTTTCTCAT (SEQ ID NO: 394)EGFR T790M PAMplification F primer (5 nt extension)TCACCTCCACCGTGTTTCTCATC (SEQ ID NO: 395)EGFR T790M PAMplification F primer (6 nt extension)TCACCTCCACCGTGTTTCTCATCA (SEQ ID NO: 396)EGFR T790M R primer (SEQ ID NO: 397) GGAGCCAATATTGTCTTTGTGTTCCCEGFR T790M Blocking primer (SEQ ID NO: 398) TCATCACGCAGCTCATGC/3Phos/

TABLE 17 RNA sequences used in this study with LbCas12a. DescriptionSequence EGFR T790M C-SNP 6 mm TAATTTCTACTAAGTGTAGATCATCACGCAGCTCATGCCCTguide RNA (SEQ ID NO: 399) EGFR T790M C-SNP 7 mmTAATTTCTACTAAGTGTAGATTCATCACGCAGCTCATGCCC guide RNA (SEQ ID NO: 400)EGFR T790M C-SNP 8 mm TAATTTCTACTAAGTGTAGATCTCATCACGCAGCTCATGCCguide RNA (SEQ ID NO: 401) EGFR T790M T-SNP 6 mmTAATTTCTACTAAGTGTAGATCATCATGCAGCTCATGCCCT guide RNA (SEQ ID NO: 402)EGFR T790M T-SNP 7 mm TAATTTCTACTAAGTGTAGATTCATCATGCAGCTCATGCCCguide RNA (SEQ ID NO: 403) EGFR T790M T-SNP 8 mmTAATTTCTACTAAGTGTAGATCTCATCATGCAGCTCATGCC guide RNA (SEQ ID NO: 404)

Example 17 Amplification of EGFR SNP T790M (c.2369C>T) Using a BlockingPrimer

This example shows that a blocking primer enriches a sample for a singlenucleotide polymorphism (SNP) versus wild-type (WT) of EGFR. Theenriched EGFR SNP was the SNP T790M (c.2369C>T). The sample comprisedboth the C SNP (WT) and the T SNP (T790M) cell free DNA EGFR DNAstandards.

FIG. 63A shows how the blocking primer blocks the forward primer frombinding to the WT nucleic acid for amplification.

FIG. 63B shows how the mutation in SNP does not result in the binding ofthe blocking primer, and therefore allowing the forward primer to bindto the SNP nucleic acid for amplification.

FIG. 63C and FIG. 63D show the detection of the EGFR C SNP using aninput of 6 ng and the detection of the EGFR T SNP using an input of 6ng, respectively, after amplification using the blocking primer strategyof FIG. 64A and FIG. 64B. PAMplification and blocking primers areprovided in TABLE 18.

TABLE 18 Primers used in this Example Description Sequence (5′→3′)EGFR T790M PAMplification F primer TCACCTCCACCGTGTTTC TCATCA(6 nt extension) (SEQ ID NO: 396) EGFR T790M R primer (SEQ ID NO: 397)GGAGCCAATATTGTCTTTGTGTTCCC EGFR T790M Blocking primer (SEQ ID NO: 398)TCATCACGCAGCTCATGC/3Phos/

Example 18 Amplification of EGFR SNP T790M (c.2369C>T) Using COLD-PCR

This example shows that a COLD-PCR enriches a sample for a singlenucleotide polymorphism (SNP) versus wild-type (WT) of EGFR. Theenriched EGFR SNP was the SNP T790M (c.2369C>T). The sample comprisedboth the C SNP (WT) and the T SNP (T790M) cell free DNA EGFR DNAstandards.

FIG. 64A shows an exemplary full COLD-PCR strategy for enriching for amutation, such as an EGFR SNP T790M (c.2369C>T).

FIG. 64B shows an exemplary full COLD-PCR strategy for enriching for amutation, such as an EGFR SNP T790M (c.2369C>T).

FIG. 65A shows the detection of the EGFR C SNP using an input of 6 ngand a crRNA corresponding to SEQ ID NO: 423 and LbCas12a (SEQ ID NO: 1)after amplification using COLD-PCR. FIG. 65B shows the detection of theEGFR C SNP using an input of 6 ng the detection of the EGFR T SNP usingan input of 6 ng and a crRNA corresponding to SEQ ID NO: 439 andLbCas12a (SEQ ID NO: 1) after amplification using COLD-PCR. COLD-PCR wasperformed using primers corresponding to SEQ ID NO: 396 and SEQ ID NO:397.

Example 19 Detection of the EGFR SNP L858R (c. 573T>G)

This example shows that that Cas12a can be used to detect a singlenucleotide polymorphism (SNP) versus wild-type (WT) of EGFR. The EGFRSNP detected was the SNP L858R (c.2573T>G). The sample comprisedsynthetic EGFR DNA standards for both WT EGFR allele with a T atposition 2573 and the G SNP (a T to G missense substitution at position2573 in the L858R locus).

FIG. 66A-FIG. 66B shows Cas12a (SEQ ID NO: 1) can detect down to 0.1-1%minor allele frequency (MAF) of EGFR L858R G SNP allele in mock cfDNAsamples (Horizon Discovery), with 1 ng total DNA input and a COLD-PCRpre-amplification step. Detection of mutant (FIG. 66A) and WT (FIG. 66B)alleles at t=40 min with low frequency EGFR standards. FIG. 66A showsdetection of the mutant allele using a gRNA corresponding to SEQ ID NO:430 and FIG. 66B shows detection of the WT allele using a gRNAcorresponding to SEQ ID NO: 429. n=3 replicates, two-tailed Student'st-test; *p<0.05, **p<0.01; bars represent mean plus SD. Target sequenceswere amplified using primers corresponding to SEQ ID NO: 450 and SEQ IDNO: 451. TABLE 19 shows the amino acid mutations (AA mutation), thechange that has occurred in the nucleotide sequence (CDS mutation), thetype of mutation, and the guide nucleic acid CRISPR RNA (crRNA) sequenceused to detect the mutation.

TABLE 19 Guide for Detecting Various Mutations EGFR COS Spacer LbCas12aMutation CDS AA MIC sequence Spacer LbCas12a crRNA Exon Group mutationmutation Type Start End ID Notes (RC) sequence crRNA handle: Exon Exon19WT TTAAG GGAGA TAATTTC R0292 19 (WT) AGAAG TGTTG TACTAA EGFR CAACACTTCTC GTGTAG Ex19 WT TCTCC TTAA ATGGAG (SEQ ID (SEQ ID ATGTTGC NO: 661)NO: 688) TTCTCTT AA (SEQ ID NO: 718) Ex19Del c.2240_2251del12p.L747_T751>S Complex_deletion_inframe 55174777 55174788  6210 GCTATGGAGA TAATTTC R0293 (28 targets CAAGG TGATT TACTAA EGFR from AATCA CCTTGGTGTAG Ex19 Roche TCTCC ATAGC ATGGAG var1 cobas test) (SEQ ID (SEQ IDATGATTC NO: 662) NO: 689) CTTGATA GC (SEQ ID NO: 719) c.2239_2247delp.L747_E749del Deletion_In_frame 55174776 55174784 6218 ATCAA GGAGATAATTTC R0294 TTAAGAGAA LRE GGAAG TGTTG TACTAA EGFR CAACA CTTCCT GTGTAGEx19 TCTCC TGAT ATGGAG var2 (SEQ ID (SEQ ID ATGTTGC NO: 663) NO: 690)TTCCTTG AT (SEQ ID NO: 720) c.2238_2255del18 p.E746_S752>DComplex_deletion_inframe 55174775 55174792 6220 CCCGT GGATC TAATTTCR0295 CGCTA CTTGA TACTAA EGFR TCAAG TAGCG GTGTAG Ex19 GATCC ACGGG ATGGATvar3 (SEQ ID (SEQ ID CCTTGAT NO: 664) NO: 691) AGCGAC GGG (SEQ ID NO:721) c.2235_2249del15 p.E746_A750delEL Deletion_In_frame 5517477255174786 6223 GTCGC GGAGA TAATTTC R0296 REA TATCA TGTTTT TACTAA EGFR(SEQ ID AAACA GATAG GTGTAG Ex19 NO: 759) TCTCC CGAC ATGGAG var4 (SEQ ID(SEQ ID ATGTTTT NO: 665) NO: 692) GATAGC GAC (SEQ ID NO: 722)c.2236_2250del15 p.E746_A750delEL Deletion_In_frame 55174773 551747876225 GTCGC GGAGA TAATTTC R0297 REA TATCA TGTCTT TACTAA EGFR (SEQ IDAGACA GATAG GTGTAG Ex19 NO: 759) TCTCC CGAC ATGGAG var5 (SEQ ID (SEQ IDATGTCTT NO: 666) NO: 693) GATAGC GAC (SEQ ID NO: 723) c.2239_2256del18p.L747_S752del Deletion_In_frame 55174776 55174793 6255 CCCGT GGTTCTAATTTC R0298 LREATS CGCTA CTTGA TACTAA EGFR (SEQ ID TCAAG TAGCG GTGTAGEx19 NO: 760) GAACC ACGGG ATGGTTC var6 (SEQ ID (SEQ ID CTTGATA NO: 667)NO: 694)  GCGACG GG (SEQ ID NO: 724) c.2237_2254del18 p.E746_S752>AComplex_deletion_inframe 55174774 55174791 12367 CCCGT GGAGC TAATTTCR0299 CGCTA CTTGA TACTAA EGFR TCAAG TAGCG GTGTAG Ex19 GCTCC ACGGG ATGGAGvar7 (SEQ ID (SEQ ID CCTTGAT NO: 668) NO: 695)  AGCGAC GGG (SEQ ID NO:725) c.2240_2254del15 p.L747_T751delLR Deletion_In_frame 5517477755174791 12369 GTCGC GGAGA TAATTTC R0300 EAT TATCA TTCCTT TACTAA EGFR(SEQ ID AGGAA GATAG GTGTAG Ex19 NO: 761) TCTCC CGAC ATGGAG var8 (SEQ ID(SEQ ID ATTCCTT NO: 669) NO: 696)  GATAGC GAC (SEQ ID NO: 726)c.2240_2257del18 p.L747_P753>S Complex_deletion_inframe 5517477755174794 12370 CCCGT GATTC TAATTTC R0301 CGCTA CTTGA TACTAA EGFR TCAAGTAGCG GTGTAG Ex19 GAATC ACGGG ATGATTC var9 (SEQ ID (SEQ ID CTTGATANO: 670) NO: 697) GCGACG GG (SEQ ID NO: 727) c.2239_2248TTAAp.L747_A750>P Complex_deletion_inframe 55174776 55174785 12382 ATCAAGGAGA TAATTTC R0302 GAGAAG>C GGAAC TGTTG TACTAA EGFR (SEQ ID CAACA GTTCCGTGTAG Ex19 NO: 762) TCTCC TTGAT ATGGAG var10 (SEQ ID (SEQ ID ATGTTGGNO: 671) NO: 698) TTCCTTG AT (SEQ ID NO: 728) c.2239_2251>Cp.L747_T751>P Complex_deletion_inframe 55174776 55174788 12383 GCTATGGAGA TAATTTC R0303 CAAGG TGGTT TACTAA EGFR AACCA CCTTG GTGTAG Ex19TCTCC ATAGC ATGGAG var11 (SEQ ID (SEQ ID ATGGTTC NO: 672) NO: 699)CTTGATA GC (SEQ ID NO: 729) c.2237_2255>T p.E746_S752>VComplex_deletion_inframe 55174774 55174792 12384 CCCGT GGAAC TAATTTCR0304 CGCTA CTTGA TACTAA EGFR TCAAG TAGCG GTGTAG Ex19  GTTCC ACGGGATGGAA var12 (SEQ ID (SEQ ID CCTTGAT NO: 673) NO: 700) AGCGAC GGG (SEQID NO: 730) c.2235_2255>AAT p.E746_S752>I Complex_deletion_inframe55174772 55174792 12385 CCCGT GGAAT TAATTTC R0305 CGCTA TTTGA TACTAAEGFR TCAAA TAGCG GTGTAG Ex19  ATTCC ACGGG ATGGAA var13 (SEQ ID (SEQ IDTTTTGAT NO: 674) NO: 701) AGCGAC GGG (SEQ ID NO: 731) c.2237_2252>Tp.E746_T751>V Complex_deletion_inframe 55174774 55174789 12386 GTCGCGGAGA TAATTTC R0306 TATCA TACCT TACTAA EGFR AGGTA TGATA GTGTAG Ex19TCTCC GCGAC ATGGAG var14 (SEQ ID (SEQ ID ATACCTT NO: 675) NO: 702)GATAGC GAC (SEQ ID NO: 732) c.2239_2258>CA p.L747_P753>QComplex_deletion_inframe 55174776 55174795 12387 CCCGT TGTTC TAATTTCR0307 CGCTA CTTGA TACTAA EGFR TCAAG TAGCG GTGTAG Ex19  GAACA ACGGGATTGTTC var15 (SEQ ID (SEQ ID CTTGATA NO: 676) NO: 703) GCGACG GG (SEQID NO: 733) c.2239_2256>CAA p.L747_S752>Q Complex_deletion_inframe55174776 55174793 12403 GTCGC GGTTG TAATTTC R0308 TATCA TTCCTT TACTAAEGFR AGGAA GATAG GTGTAG Ex19  CAACC CGAC ATGGTTG var16 (SEQ ID (SEQ IDTTCCTTG NO: 677) NO: 704) ATAGCG AC (SEQ ID NO: 734) c.2237_2253>TTGp.E746_T751>VA Complex_deletion_inframe 55174774 55174790 12416 GCTATGGAGA TAATTTC R0309 CT CAAGG AGCAA TACTAA EGFR TTGCTT CCTTG GTGTAG Ex19 CTCC ATAGC ATGGAG var17 (SEQ ID (SEQ ID AAGCAA NO: 678) NO: 705) CCTTGATAGC (SEQ ID NO: 735) c.2238_2252>GCA p.L747_T751>QComplex_deletion_inframe 55174775 55174789 12419 GCTAT GGAGA TAATTTCR0310 CAAGG TTGCT TACTAA EGFR AGCAA CCTTG GTGTAG Ex19  TCTCC ATAGCATGGAG var18 (SEQ ID (SEQ ID ATTGCTC NO: 679) NO: 706) CTTGATA GC (SEQID NO: 736) c.2238_2248>GC p.L747_A750>P Complex_deletion_inframe55174775 55174785 12422 ATCAA GGAGA TAATTTC R0311 GGAGC TGTTG TACTAAEGFR CAACA GCTCC GTGTAG Ex19 TCTCC TTGAT ATGGAG var19 (SEQ ID (SEQ IDATGTTGG NO: 680) NO: 707) CTCCTTG AT (SEQ ID NO: 737) c.2237_2251del15p.E746_T751>A Complex_deletion_inframe 55174774 55174788 12678 GTCGCGGAGA TAATTTC R0312 TATCA TGCCT TACTAA EGFR AGGCA TGATA GTGTAG Ex19 TCTCC GCGAC ATGGAG var20 (SEQ ID (SEQ ID ATGCCTT NO: 681) NO: 708)GATAGC GAC (SEQ ID NO: 738) c.2236_2253del18 p.E746_T751delDeletion_In_frame 55174773 55174790 12728 CCCGT GGAGA TAATTTC R0313ELREAT CGCTA CTTGA TACTAA EGFR (SEQ ID TCAAG TAGCG GTGTAG Ex19  NO: 763)TCTCC ACGGG ATGGAG var21 (SEQ ID (SEQ ID ACTTGAT NO: 682) NO: 709)AGCGAC GGG (SEQ ID NO: 739) c.2235_2248>AAT p.E746_A750>IPComplex_deletion_inframe 55174772 55174785 13550 ATCAA GGAGA TAATTTCR0314 TC AATTC TGTTG TACTAA EGFR CAACA GAATT GTGTAG Ex19  TCTCC TTGATATGGAG var22 (SEQ ID (SEQ ID ATGTTGG NO: 683) NO: 710) AATTTTG AT (SEQID NO: 740) c.2235_2252>AAT p.E746_T751>I Complex_deletion_inframe55174772 55174789 13551 GTCGC GGAGA TAATTTC R0315 TATCA TATTTT TACTAAEGFR AAATA GATAG GTGTAG Ex19  TCTCC CGAC ATGGAG var23 (SEQ ID (SEQ IDATATTTT NO: 684) NO: 711) GATAGC GAC (SEQ ID NO: 741) c.2235_2251>AATp.E746_T751>IP Complex_deletion_inframe 55174772 55174788 13552 GCTATGGAGA TAATTTC R0316 TC CAAAA TGGAA TACTAA EGFR TTCCA TTTTG GTGTAG Ex19 TCTCC ATAGC ATGGAG var24 (SEQ ID (SEQ ID ATGGAA NO: 685) NO: 712)TTTTGAT AGC (SEQ ID NO: 742) c.2237_2257>TCT p.E746_P753>VSComplex_deletion_inframe 55174774 55174794 18427 CCCGT GAGAC TAATTTCR0317 CGCTA CTTGA TACTAA EGFR TCAAG TAGCG GTGTAG Ex19  GTCTC ACGGGATGAGA var25 (SEQ ID (SEQ ID CCTTGAT NO: 686) NO: 713) AGCGAC GGG (SEQID NO: 743) c.2233_2247del15 p.K745_E749delKE Deletion_In_frame 5517477055174784 26038 GTCGC GGAGA TAATTTC R0319 LRE TATCG TGTTG TACTAA EGFR(SEQ ID CAACA CGATA GTGTAG Ex19  NO: 764) TCTCC GCGAC ATGGAG var27(SEQ ID (SEQ ID ATGTTGC NO: 687) NO: 714) GATAGC GAC (SEQ ID NO: 744)Exon T790 WT Requires 20 (WT) PAMplifi- cation T790M c.2369C>T p.T790MSubstitution_Missense 55181378 55181378 6240 Requires PAMplifi- cationExon L858 WT GGCTG TAATTTC R0435 21 (WT) GCCAA TACTAA EGFR ACTGC GTGTAGL858 WT TGGGT ATGGCT (SEQ ID GGCCAA NO: 715) ACTGCTG GGT (SEQ ID NO:745) L858R c.2573T>G p.L858R Substitution_Missense 55191822 551918226224 GGCGG TAATTTC R0436 GCCAA TACTAA EGFR ACTGC GTGTAG L858R TGGGTATGGCG (G-SNP) (SEQ ID GGCCAA NO: 716) ACTGCTG GGT (SEQ ID NO: 746)c.2573_2574TG>GT p.L858R Substitution_Missense 55191822 55191823 12429GGCGT TAATTTC R0437 GCCAA TACTAA EGFR ACTGC GTGTAG L858R TGGGT ATGGCG(GT-SNP) (SEQ ID TGCCAA NO: 717) ACTGCTG GGT (SEQ ID NO: 747)

Example 20 Assessment of Guide RNAs for Detection of EGFR-Exon 19Deletions

This example shows that Cas12a can be used to detect deletions in EGFR.The deletions detected were located in exon 19. Twenty-six guide RNAswere designed to detect deletions in exon 19 of the EGFR DNA sequence.

26 guides (SEQ ID NO: 481-SEQ ID NO: 506, shown in FIG. 67 as “Var”)were designed and compared to a wild-type guide (SEQ ID NO: 480). Theremaining 26 guides were used to screen 1 nM synthetic DNA twistfragments. Guide sequence are provided in TABLE 20.

TABLE 20 Wild Type and Variant gRNAs for Detection of Exon 19 DeletionsSEQ ID NO: Variant gRNA Sequence SEQ ID EGFRUAAUUUCUACUAAGUGUAGAUGGAGAUGUUGCUUCUCUUAA NO: 480 Exon19 WT SEQ ID EGFRUAAUUUCUACUAAGUGUAGAUGGAGAUGAUUCCUUGAUAGC NO: 481 Exon19 var01 SEQ IDEGFR UAAUUUCUACUAAGUGUAGAUGGAGAUGUUGCUUCCUUGAU NO: 482 Exon19 var02SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGGAUCCUUGAUAGCGACGGG NO: 483 Exon19var03 SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGGAGAUGUUUUGAUAGCGAC NO: 484Exon19 var04 SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGGAGAUGUCUUGAUAGCGACNO: 485 Exon19 var05 SEQ ID EGFRUAAUUUCUACUAAGUGUAGAUGGUUCCUUGAUAGCGACGGG NO: 486 Exon19 var06 SEQ IDEGFR UAAUUUCUACUAAGUGUAGAUGGAGCCUUGAUAGCGACGGG NO: 487 Exon19 var07SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGGAGAUUCCUUGAUAGCGAC NO: 488 Exon19var08 SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGAUUCCUUGAUAGCGACGGG NO: 489Exon19 var09 SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGGAGAUGUUGGUUCCUUGAUNO: 490 Exon19 var10 SEQ ID EGFRUAAUUUCUACUAAGUGUAGAUGGAGAUGGUUCCUUGAUAGC NO: 491 Exon19 var11 SEQ IDEGFR UAAUUUCUACUAAGUGUAGAUGGAACCUUGAUAGCGACGGG NO: 492 Exon19 var12SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGGAAUUUUGAUAGCGACGGG NO: 493 Exon19var13 SEQ ID R306 EGFR UAAUUUCUACUAAGUGUAGAUGGAGAUACCUUGAUAGCGAC NO: 494Exon19 var14 SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUUGUUCCUUGAUAGCGACGGGNO: 495 Exon19 var15 SEQ ID EGFRUAAUUUCUACUAAGUGUAGAUGGUUGUUCCUUGAUAGCGAC NO: 496 Exon19 var16 SEQ IDEGFR UAAUUUCUACUAAGUGUAGAUGGAGAAGCAACCUUGAUAGC NO: 497 Exon19 var17SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGGAGAUUGCUCCUUGAUAGC NO: 498 Exon19var18 SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGGAGAUGUUGGCUCCUUGAU NO: 499Exon19 var19 SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGGAGAUGCCUUGAUAGCGACNO: 500 Exon19 var20 SEQ ID EGFRUAAUUUCUACUAAGUGUAGAUGGAGACUUGAUAGCGACGGG NO: 501 Exon19 var21 SEQ IDEGFR UAAUUUCUACUAAGUGUAGAUGGAGAUGUUGGAAUUUUGAU NO: 502 Exon19 var22SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGGAGAUAUUUUGAUAGCGAC NO: 503 Exon19var23 SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGGAGAUGGAAUUUUGAUAGC NO: 504Exon19 var24 SEQ ID EGFR UAAUUUCUACUAAGUGUAGAUGAGACCUUGAUAGCGACGGGNO: 505 Exon19 var25 SEQ ID EGFRUAAUUUCUACUAAGUGUAGAUGGAGAUUCCUUGAUAGCGAC NO: 488 Exon19 var26 SEQ IDEGFR UAAUUUCUACUAAGUGUAGAUGGAGAUGUUGCGAUAGCGAC NO: 506 Exon19 var27

Resulting signals were measured using DNA Endonuclease Targeted CRISPRTrans Reporter (DETECTR) techniques. Two guide sequences (SEQ ID NO: 493and SEQ ID NO: 499) showed similar detection sensitivity to wild-type(FIG. 67). The other 24 guides showed activity greater than wild-type,with three variants (SEQ ID NO: 485, SEQ ID NO: 488, and SEQ ID NO: 490)showing the highest detection sensitivity. Targets corresponding to SEQID NO: 452-SEQ ID NO: 477 and SEQ ID NO: 479, provided in TABLE 21, weredetected.

TABLE 21 EGFR Exon 19 Deletions Wild Type and Variant SequencesSEQ ID NO: Variant gRNA Sequence SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 452 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var01CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATCATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 453 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var02CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGT G SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 454 Exon19- GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var03CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGATCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 455 Exon19- GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var04CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 456 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var05CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 457 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var06CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAACCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 458 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var07CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 459 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var08CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 460 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var09CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 461 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var10CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAACCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGT G SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 462 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var11CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAACCATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 463 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var12CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGTTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 464 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var13CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAAATTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 465 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var14CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGTATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 466 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var15CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAACAGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 467 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var16CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAACAACCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 468 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var17CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGTTGCTTCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 469 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var18CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAGCAATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 470 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var19CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAGCCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGT G SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 471 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var20CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGCATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 472 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var21CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGTCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 473 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var22CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAAATTCCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 474 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var23CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAAATATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 475 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var24CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAAATTCCATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 476 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var25CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGTCTCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 477 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var27CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 478 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG var28CACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGGAGGCTCAGTG SEQ ID NO: EGFR-GAAGTGCCATTCCGCCTGACCTAGCCCCAGTGTCCCTCACCTTCGG 479 Exon19-GGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGG WTCACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTAGGCTAGGTGG AGGCTCAGTG

FIG. 67 shows a heat map of the DETECTR assays for each of the 26 guidevariants (“Var”) and a wild type (“WT”) control tested. Fluorescence isthe output of the DETECTR assay, and indicates that a Cas12aprogrammable nuclease (SEQ ID NO: 1) was activated by a target DNA tocollaterally cleave a fluorescently labeled reporter, and is, thus, ameasure of variant detection sensitivity. Guide variants detected theEGFR sequence comprising the deletion, while the WT control detected theEGFR wild type sequence. Screening was performed in the presence of 1 nMsynthetic DNA twist fragments.

TABLE 19 shows the amino acid mutations (AA mutation), the change thathas occurred in the nucleotide sequence (CDS mutation), the type ofmutation, the start and end nucleotide positions of the deletion on thecorresponding chromosome, and the guide nucleic acid CRISPR RNA (crRNA)sequence used to detect the mutation.

Example 21 PAM Forward Primer (PAMplification Primer)

This example shows the optimal PAM forward primer (also referred to as aPAMplification primer) for use in amplifying a target nucleic acid tocomprise a sequence encoding a PAM.

FIG. 68A-FIG. 68B and FIG. 69A-FIG. 69B shows the PAM forward primer(also referred to as a PAMplification primer). The single nucleotidemismatch was anchored at positions 3-8 or 5-8 nt downstream of the PAM/.PAMplification primers with 2 nt or 4 nt extensions at the 3′ end weretested for their ability to discriminate the non-cognate targetcontaining a single nucleotide mismatch/polymorphism (SNP). Here, a 4 ntPAMplification 3′ extension is better at SNP detection compared to the 2nt extension. The mismatch position is optimal around positions 6, 7 or8. Primers used in this assay are provided in TABLE 22.

Example 22 Cas12 Recognizes dU-Containing PAM and Target Nucleic Acids

This example shows that Cas12 recognizes dU-containing PAM. Furthermore,a Cas12 recognizes target nucleic acids comprising dU.

FIG. 70A-FIG. 70B shows that Cas12 recognizes dU-containing PAM andtarget sequences from 100 nM to 10 pM. FIG. 70A: WT SNP-targeting guideRNA; FIG. 70B: mutant SNP-targeting guide RNA. Left to right for bothFIG. 70A and FIG. 70B: (top left) WT sequence with dT-containing target,(top middle) mutant sequence with dT-containing target, (top right)mutant sequence with dU-containing PAM and target, (bottom left) notarget, (bottom right) mutant sequence with dT-containing PAM anddU-containing target. Cas12 is capable of SNP detection withdU-containing sequences (both PAM and target) without compromisingsensitivity. Primers used in this assay are provided in TABLE 22.

Example 23 Cas12 Recognizes dU-Containing Amplicons of the ALDH2 WTAllele

This example shows that Cas 12 recognizes a dU-containing amplicons ofthe ALDH2 WT allele. Additionally, the Cas12 was able to distinguishdU-containing amplicons of the ALDH2 WT allele and dU-containingamplicons of the ALDH2 SNP allele.

FIG. 71A-FIG. 71B shows the detection of ALDH2 WT allele from humangenomic DNA (SEQ ID NO: 417) with dU-containing amplicons with Cas12.The sequence of the target is shown in FIG. 71A—the top strand has asequence of5′-CACACTCACAGTTTTCACTTCAGTGTATGCCTGCAGCCCGTACTCGCCCAACTCC-3′ (SEQ IDNO: 752) and the bottom strand has a sequence of5′-GGAGTTGGGCGAGTACGGGCTGCAGGCATACACTGAAGTGAAAACTGTGAGTGTG-3′ (SEQ IDNO: 753). The ALDH2 gene was amplified from human saliva containing theWT allele using Taq master mix containing dUTP in place of dTTP, suchthat all T nucleotides with the above annotated ALDH2 target sequencehas been replaced by U nucleotides. The amplicon was added directly to aCas12 DETECTR assay. Cas12 guide RNAs targeting the ALDH2 WT alleledetected only the cognate WT sequence and not the mutant allele,demonstrating that Cas12 is capable of SNP detection with dU-containingtargets.

Example 24 Cas12 Recognizes dU-Containing Amplicons of at a LowFrequency

This example shows that Cas 12 recognizes a dU-containing amplicons at alow frequency.

FIG. 58A-FIG. 58C show the PAMplification primer produces dU-containingamplicons for detection of mutant sequences at low frequency. Cas12guide RNAs were designed to target the T790M mutant allele (c.2369C>T,at guide mismatch position 7) in Horizon Discovery EGFR cfDNA standardsat 0-5% minor allele frequencies (MAF) with 2 ng input DNA.PAMplification primers include 4-6 nt extensions at the 3′ enddownstream of the embedded PAM. n=3 technical replicates; bars representmean±SD.

FIG. 60A-FIG. 60C show the detection of low frequency SNPs usingPAMplification with 6 nt extension and dU-containing amplicons. Cas12acan detect down to 0.1-1% minor allele frequency (MAF) of EGFR T790M inmock cfDNA samples (Horizon Discovery), with 2 ng total DNA input. n=3replicates, two-tailed Student's t-test; *p<0.05, **p<0.01, ***p<0.001,****p<0.0001; bars represent mean plus SD.

TABLE 22 Primers used in Examples 21, 22, and 24 DescriptionSequence (5′ → 3′) T790M dUTP  CTCCACCGTGC/ideoxyU//ideoxyU// PAM 6 mmideoxyU/GCA/ideoxyU/CACGCAGC/ NTS  ideoxyU/CA/ideoxyU/GCCC/ (SEQ ID ideoxyU//ideoxyU/ NO: 408) TCGGCGCCTCCTGGACTAT T790M dUTP ATAGTCCAGGAGGCAGCCGAAGGGCA/ PAM 6mm ideoxyU/GAGC/ideoxyU/GCG/ TS ideoxyU/GA/ideoxyU/ (SEQ ID  GCAAAGCACGGTGGAG NO: 409) T790M dUTP CTCCACCGTGCTTTGCA/ideoxyU/ 6mm NTS CACGCAGC/ideoxyU/CA/ideoxyU/ (SEQ ID GCCC/ideoxyU//ideoxyU/ NO: 410) TCGGCGCCTCCTGGACTAT T790M dUTP ATAGTCCAGGAGGCAGCCGAAGGGCA/ 6mm TS ideoxyU/GAGC/ideoxyU/GCG/ (SEQ ID ideoxyU/GA/ideoxyU/ NO: 411) GCAAAGCACGGTGGAG EGFR T790M  TCACCTCCACCGTGTTTC TCAT 7mm PAM 4nt_F  (SEQ ID NO: 394) EGFR T790M  TCACCTCCACCGTGTTTC TCATC 7mm PAM 5nt_F  (SEQ ID NO: 395) EGFR T790M  TCACCTCCACCGTGTTTC TCATCA 7mm PAM 6nt_F  (SEQ ID NO: 396) EGFR T790M GGAGCCAATATTGTCTTTGTGTTCCC std R (SEQ ID  NO: 397)

Example 25 Ratio of LAMP Amplicon in Cas12 Detection Reaction

This example describes ratios of LAMP amplicon used in Cas12 detectionreactions provided herein. A detection assay using a Cas12 variant (SEQID NO: 11) was performed in the presence of increasing amounts of LAMPamplified genomic DNA target nucleic acid sequence. The target nucleicacid sequence was amplified for 30 minutes at 60° C. using LAMPamplification. Increasing volumes of the amplified nucleic acid sequencewere combined in a 20 μL Cas12 detection reaction. The Cas12 detectionassay was run for 30 minutes at 37° C.

FIG. 72 shows detection of amplified HERC2 genomic DNA using a Cas12variant (SEQ ID NO: 11) in the presence of increasing amounts of LAMPamplified DNA (“LAMP.Amplicon”). The HERC2 target was amplified fromHeLa genomic DNA using LAMP amplification with the HERC2 LAMP primersshown in TABLE 14 (SEQ ID NO: 233-SEQ ID NO: 238). Each detectionreaction was performed in the presence of 1 μL to 14 μL LAMP amplifiedDNA in 20 μL reactions. A negative control reaction was performedwithout LAMP amplified DNA (0 μL). Detection of the LAMP amplified DNAwas quantified by fluorescence upon cleavage of a reporter (SEQ ID NO:119 with N-terminal /5Alex594N/ and C-terminal /3IAbRQSp/) by anactivated Cas12 programmable nuclease upon binding of a guide RNA to thetarget LAMP amplified DNA.

The results indicated that the performance of the Cas12 detection assaywas stable at 1 μL of LAMP amplified DNA in 20 μL reaction volumes up to1 μL LAMP amplified DNA in 20 μL reaction volumes. Increasing the ratioto 12 μL, 13 μL, or 14 μL of LAMP amplicon in 20 μL reaction volumes,led to a decrease in assay performance.

Example 26 Addition of an Artificial PAM to LAMP FIP or BIP Primers

This example describes addition of an artificial PAM to LAMP FIP or BIPprimers as described herein. An artificial PAM was added to a targetnucleic sequence by LAMP amplifying the target nucleic acid using a FIPor BIP primer with the artificial PAM sequence. The PAM was inserted atdifferent positions using different FIP primers shown in TABLE 23, withthe PAM indicated by bold and underlining. This method of PAMintroduction using LAMP amplification (referred to herein asPAMplification) was used to generate a target site for a CRISPR/Cassystem that would not have otherwise been accessible.

TABLE 23 Artificial PAM Position within LAMP Amplification FIP PrimersSEQ ID PAM NO: Position Sequence SEQ ID No PAMCGCCTCTTGGATCAGACACATGTGTTAATA NO: 235 CAAAGGTACAGGA SEQ ID PositionCAAA TCTTGGATCAGACACATGTGTTAATA NO: 265 1 CAAAGGTACAGGA SEQ ID PositionC CAAA CTTGGATCAGACACATGTGTTAATA NO: 266 2 CAAAGGTACAGGA SEQ ID PositionCG CAAA TTGGATCAGACACATGTGTTAATA NO: 267 3 CAAAGGTACAGGA SEQ ID PositionCGC CAAA TGGATCAGACACATGTGTTAATA NO: 268 4 CAAAGGTACAGGA SEQ ID PositionCGCC CAAA GGATCAGACACATGTGTTAATA NO: 269 5 CAAAGGTACAGGA SEQ ID PositionCGCCT CAAA GATCAGACACATGTGTTAATA NO: 270 6 CAAAGGTACAGGA SEQ ID PositionCGCCTC CAAA ATCAGACACATGTGTTAATA NO: 271 7 CAAAGGTACAGGA SEQ ID PositionCGCCTCT CAAA TCAGACACATGTGTTAATA NO: 272 8 CAAAGGTACAGGA SEQ ID PositionCGCCTCTT CAAA CAGACACATGTGTTAATA NO: 273 9 CAAAGGTACAGGA SEQ ID PositionCGCCTCTTG CAAA AGACACATGTGTTAATA NO: 274 10 CAAAGGTACAGGA SEQ IDPosition CGCCTCTTGG CAAA GACACATGTGTTAATA NO: 275 11 CAAAGGTACAGGASEQ ID Position CGCCTCTTGGA CAAA ACACATGTGTTAATA NO: 276 12CAAAGGTACAGGA SEQ ID Position CGCCTCTTGGAT CAAA CACATGTGTTAATA NO: 27713 CAAAGGTACAGGA SEQ ID Position CGCCTCTTGGATC CAAA ACATGTGTTAATANO: 278 14 CAAAGGTACAGGA SEQ ID Position CGCCTCTTGGATCA CAAACATGTGTTAATA NO: 279 15 CAAAGGTACAGGA SEQ ID Position CGCCTCTTGGATCAGCAAA ATGTGTTAATA NO: 280 16 CAAAGGTACAGGA SEQ ID PositionCGCCTCTTGGATCAGA CAAA TGTGTTAATA NO: 281 17 CAAAGGTACAGGA

FIG. 73 shows a schematic of addition of an artificial PAM to LAMP FIPor BIP primers. PAMs were introduced at different positions within theLAMP primer, and gRNAs were designed relative to each PAM for use inCRISPR-based detection assays of target nucleic acids. The PAM wasintroduced at different positions within the LAMP FIP primer, and thetarget nucleic acid was detected with gRNAs for each PAM position toassess the impact of PAM placement in an FIP primer on (1) theefficiency of LAMP amplification and (2) non-specific activation oftrans cleavage by the primer binding to the gRNA-Cas protein complex.

FIG. 74 shows LAMP amplification of a target human genomic DNA (HERC2,SEQ ID NO: 416) with an FIP primer having PAM sequences at varyingpositions to introduce an artificial PAM in the HERC2 target nucleicacid. PAM introduction with the FIP primer and gRNA binding sites foreach corresponding PAM containing FIP primer are shown in FIG. 73. Forexample, an FIP primer having the PAM sequence at position 17 (17^(th)nucleotide from the 5′ end of the FIP primer; depicted as “PAM Pos 17”)is used with a gRNA sequence for Pos 17 (5′ end of gRNA is adjacent tothe 5′ end of the PAM sequence in the primer; depicted as “gRNA seq forPos 17”). The target was amplified using primers corresponding to SEQ IDNO: 233-SEQ ID NO: 234 and SEQ ID NO: 236-SEQ ID NO: 238 with a variableFIP depending on the position of the artificially introduced PAM. FIPscorresponding to SEQ ID NO: 265-SEQ ID NO: 281 were used to insertartificial PAMs at position 1-position 17, respectively. The FIPcorresponding to SEQ ID NO: 235 was used to amplify the target withoutintroducing a PAM. Amplification was monitored using a SYTO9 DNA bindingdye. Rate of amplification was quantified by the time to result, whichwas determined by the time to reach half maximum SYTO9 fluorescenceintensity. Time to result was indicative of the time to reachexponential amplification. A lower time to result value indicated fasteramplification. The results demonstrated that positioning the PAMsequence near the 5′ end of the LAMP FIP primer led to sloweramplification compared to the control FIP primer lacking a PAM. Mostadded PAM sequences positioned near the center of the FIP primer (fromabout position 6 to about position 15) showed similar amplificationtimes compared to the control.

A HERC2 target nucleic acid with artificially inserted PAM sequences atvarious positions (position 1 to position 17) within the target nucleicacid were detected using a Cas12 detection assay. The HERC2 target wasamplified using LAMP primers SEQ ID NO: 233, SEQ ID NO: 234, and SEQ IDNO: 235-SEQ ID NO: 238. IP primers corresponding to SEQ ID NO: 265-SEQID NO: 281 were used to introduce artificial PAs at position 1-position17, respectively. The FIP primer corresponding to SEQ ID NO: 235 wasused to amplify the target without inserting an artificial PAM. gRNAswere designed to hybridize to the target nucleic acid sequence with thePAM sequence inserted at various positions. FIG. 75 shows detection of atarget nucleic acid with an artificially introduced PAM using a Cas2variant (SEQ ID NO: 11). gRNAs corresponding to SEQ ID NO: 283-SEQ IDNO: 299 were used to detect target nucleic acids with artificiallyintroduced PAs at position 1-position 17, respectively. Sequences of thegRNAs are provided in TABLE 24. Artificial PAMs were introduced atdifferent positions of a FIP primer, as illustrated in FIG. 73. Uponhybridization of the gRNA to the target, SEQ ID NO: 11 was activated andcleaved reporters (SEQ ID NO: 119 with N-terminal /5Alex594N/ andC-terminal /3IAbRQSp/), releasing a fluorescent detectable signal. Thus,target nucleic acids were detected by measuring fluorescence.Fluorescence was measured following LAMP with genomic DNA, LAMP with notarget (negative control), or a water negative control (“water controlfor detection assay”). The detection assay was performed at 37° C. for90 minutes using 1 μL of the LAMP amplicon per 20 μL reaction.

TABLE 24 gRNAs for Detection of Target Sequences withArtifically Introdiced PAMs SEQ ID PAM NO: Position gRNA Sequence SEQ IDPosition UAAUUUCUACUAAGUGUAGAUTGCTCAAAT NO: 283 1 GAAACTGGCCT SEQ IDPosition UAAUUUCUACUAAGUGUAGAUGCTCAAATG NO: 284 2 AAACTGGCCTC SEQ IDPosition UAAUUUCUACUAAGUGUAGAUCTCAAATGA NO: 285 3 AACTGGCCTCG SEQ IDPosition UAAUUUCUACUAAGUGUAGAUTCAAATGAA NO: 286 4 ACTGGCCTCGC SEQ IDPosition UAAUUUCUACUAAGUGUAGAUCAAATGAAA NO: 287 5 CTGGCCTCGCC SEQ IDPosition UAAUUUCUACUAAGUGUAGAUAAATGAAAC NO: 288 6 TGGCCTCGCCT SEQ IDPosition UAAUUUCUACUAAGUGUAGAUAATGAAACT NO: 289 7 GGCCTCGCCTC SEQ IDPosition UAAUUUCUACUAAGUGUAGAUATGAAACTG NO: 290 8 GCCTCGCCTCT SEQ IDPosition UAAUUUCUACUAAGUGUAGAUTGAAACTGG NO: 291 9 CCTCGCCTCTT SEQ IDPosition UAAUUUCUACUAAGUGUAGAUGAAACTGGC NO: 292 10 CTCGCCTCTTG SEQ IDPosition UAAUUUCUACUAAGUGUAGAUAAACTGGCC NO: 293 11 TCGCCTCTTGG SEQ IDPosition UAAUUUCUACUAAGUGUAGAUAACTGGCCT NO: 294 12 CGCCTCTTGGA SEQ IDPosition UAAUUUCUACUAAGUGUAGAUACTGGCCTC NO: 295 13 GCCTCTTGGAT SEQ IDPosition UAAUUUCUACUAAGUGUAGAUCTGGCCTCG NO: 296 14 CCTCTTGGATC SEQ IDPosition UAAUUUCUACUAAGUGUAGAUTGGCCTCGC NO: 297 15 CTCTTGGATCA SEQ IDPosition UAAUUUCUACUAAGUGUAGAUGGCCTCGCC NO: 298 16 TCTTGGATCAG SEQ IDPosition UAAUUUCUACUAAGUGUAGAUGCCTCGCCT NO: 299 17 CTTGGATCAGA

Adding an artificial PAM at various positions within a LAMP FIP primerled to non-specific activation of SEQ ID NO: 11 trans cleavage activitywhen at least 12 nucleotides overlapped between the gRNA and the LAMPFIP primer. The degree of non-specific trans cleavage activity isexpected to be impacted by the melting temperature of the overlappinggRNA and LAMP primer sequence, with a higher melting temperature leadingto more non-specific trans cleavage activity.

Based on the time to amplification shown in FIG. 74 and the Cas12detection shown in FIG. 75, the results demonstrated that artificial PAMsequences were preferably positioned away from the 5′ end of the FIP(F1c region) or BTP primer (B1c region) and towards the center (position6 to position 15) of the FIP or BIP primers. Additionally, positioningthe artificial PAM such that less than 5000 of the primer overlappedwith the gRNA sequence decreased non-specific trans cleavage activation.The assay also showed better detection sensitivity and specificity atPAM insertion positions where fewer mutations were made in the primer toinsert the artificial PAM sequence (e.g., PAMs inserted at positions 13,15, or 17 having 1 or 2 changes relative to the wild type sequence). Incontrast, detection sensitivity was lower at PAM insertion positionswhere more mutations were made in the primer to inset the artificial PAMsequence (e.g., PAMs inserted at positions 12 or 14 having 3 or 4changes relative to the wild type sequence).

Example 27 SEQ ID NO: 11 Programmable Nuclease SNP Sensitivity Along aTarget Sequence

This example describes sensitivity of a Cas12 variant programmablenuclease (SEQ ID NO: 11) to SNPs positioned along a target sequence.

In a first assay, sensitivity to point mutations in a target sequencewith a native PAM site was tested. To determine which positions along atarget nucleic acid sequence were most sensitive to single pointmutations, all four nucleotide possibilities (A, T, C, or G) at eachposition were tiled along a target of a target nucleic acid sequence.The assay was performed for two target nucleic acid sequences, a HERC2target nucleic acid sequence and an ALDH target nucleic acid sequence.Both target nucleic acid sequences comprised a native PAM site. Thetarget nucleic acids comprising the PAM site with each of all possiblepoint mutations were detected using a SEQ ID NO: 11 programmablenuclease. FIG. 76 shows detection of single point mutations at differentpositions along a nucleic acid sequence using a SEQ ID NO: 11programmable nuclease. Point mutations to each nucleic acid (A, T, C, orG, “SNP Base (target)”) were made along a target nucleic acid sequenceat different positions relative to a native PAM. To determine thesensitivity of the detection assay to single point mutations (e.g., aSNP), the target nucleic acid was detected using a gRNA directed tohybridize to the wild type sequence. Black circles label with “WT”indicate the nucleotide at each position of the wild type sequence thatis reverse complementary to the gRNA sequence. The assay was performedwith a HERC2 target sequence (top panel, wild type sequenceTCGTAATTCACAGTTCAAGA, SEQ ID NO: 416) or an ALDH target sequence (bottompanel, wild type sequence 3′-TGAAGTCACATACGGACGTC-5′, SEQ ID NO: 417).The HERC2 sequence was detected using a gRNA corresponding to SEQ ID NO:246 (top plot) and the ALDH sequence was detected using a gRNAcorresponding to SEQ ID NO: 425 (bottom plot). Upon hybridization ofgRNA to the target nucleic acid, SEQ ID NO: 11 is activated and transcleaves a reporter (SEQ ID NO: 119 with N-terminal /56-FAM/ andC-terminal /3IABkFQ/), releasing a fluorescent detectable label.Detection of target nucleic acids with SNPs was carried out by measuringfluorescence from the cleaved detectable label, and the maximum rate(“Average Max Rate”) was calculated as fluorescence units per minute andaveraged between four replicates. Results indicated that SEQ ID NO: 11was sensitive to point mutations along the entire length of the gRNAtarget site. The specificity for individual point mutations depended onsequence context of the target nucleic acid.

In a second assay, a programmable nuclease of SEQ ID NO: 11 was used todetect variants at two SNP sites in a target nucleic acid sequencewithout a native PAM. The detection assay was run for 90 minutes at 37°C. with either a wild type DNA (“WT”), a target DNA with a mutation at afirst SNP (“rs738408”), or a target DNA with a mutation at a second SNP(“rs738409”). FIG. 77 shows detection of two PNPLA3 SNPs in a targetnucleic acid sequence without a native “TTTN” PAM sequence using aprogrammable nuclease of SEQ ID NO: 11. Target nucleic acids testedcontained the wild type sequence (“WT”), a sequence with a mutation at afirst SNP (“rs738408”), a sequence with a mutation at a second SNP(“rs738409”), or a sequence with mutations at the first SNP and thesecond SNP (“rs738409/408”). Target sequences are provided in TABLE 25.

TABLE 25 PNPLA3 Target Sequences SEQ ID Target NO: Name Target SequenceSEQ PNPLA3 TGCCTGCTGACTGCTCTGTAGCACAGTGCTTCG ID rs738409 +CAAAGTGTGATCCTGGGACCAGCAGAGCAGCAG NO: rs738408CTCCTTTGAGCTTATTGGAATGGCAGACCCTCA 412 GGTCCCACCTCTGACCTGCTGCATGGGAATTCTGGGGAGGGACGCAGAATCTCTGGTTCCACAGGC TCTCCGGTGATGCTAATGAATACCGGCATTTGAACAGCACCGATCTAGCCCCTTTCAGTCCATGAG CCAACAACCCTTGGTCCTGTCTGTGGTGACCCAGTGTGACTCTCATGGGGAGCAAGGAGAGGAAGT TGAAGTTCACTGACAGGGTTGTTAAGGGGATTATGCAATAGATGAGACCCATGGGCCTGAAGTCCG AGGGTGTATGTTAGTTCCCCGTTCTTTTGACCCATGGATTAACCTACTCTGTGCAAAGGGCATTTT CAAGTTTGTTGCCCTGCTCACTTGGAGAAAGCTTATGAAGGATCAGGAAAATTAAAAGGGTGCTCT CGCCTATAACTTCTCTCTCCTTTGCTTTCACAGGCCTTGGTATGTTCCTGCTTCATGCCTTTCTAC AGTGGCCTTATCCCTCCTTCCTTCAGAGGCGTGGTAAGTCGGCTTTCTCTGCTAGCGCTGAGTCCT GGGGGCCTCTGAAGTGTGCTCACACATCTCCTGCCTGCAGGGCACTGGTGTCGGGCACCTCAGGGT CTGTCCCATGGTGGAGCCCCATGCCTCACTGCCTTTCAGACAGAGTAGCCACAGCTGGCCCTATTT CCAGGCTACCCGGGCAGCAAAACTTACTGCATGTGTAATTAATTATTTGGCTATCTGTAAGGTAAA CTGGCTGGTTCACTTAATCTGCACCTTAAGCATCAGATAGCTTCTCAGTGATCTAGTTAAACTATA TGATGTTGGCCAGGCGCGGTGGCTCATGTCTGTAATCCCAGCACTTTGGGAGCCTGAAGCAGGCAG ATCACTTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACAGTGTGAAACTCTGTCTCTCCTAAAAA TACAAAAATTAGCTGGGCATGGTGGTGTGCACCTGTAATCCCAGCTGCTCGGGAGGCTGAGGCAGG AGAATTGCTTGAACTTGGGA SEQ PNPLA3TGCCTGCTGACTGCTCTGTAGCACAGTGCTTCG ID rs738408CAAAGTGTGATCCTGGGACCAGCAGAGCAGCAG NO: CTCCTTTGAGCTTATTGGAATGGCAGACCCTCA413 GGTCCCACCTCTGACCTGCTGCATGGGAATTCT GGGGAGGGACGCAGAATCTCTGGTTCCACAGGCTCTCCGGTGATGCTAATGAATACCGGCATTTGA ACAGCACCGATCTAGCCCCTTTCAGTCCATGAGCCAACAACCCTTGGTCCTGTCTGTGGTGACCCA GTGTGACTCTCATGGGGAGCAAGGAGAGGAAGTTGAAGTTCACTGACAGGGTTGTTAAGGGGATTA TGCAATAGATGAGACCCATGGGCCTGAAGTCCGAGGGTGTATGTTAGTTCCCCGTTCTTTTGACCC ATGGATTAACCTACTCTGTGCAAAGGGCATTTTCAAGTTTGTTGCCCTGCTCACTTGGAGAAAGCT TATGAAGGATCAGGAAAATTAAAAGGGTGCTCTCGCCTATAACTTCTCTCTCCTTTGCTTTCACAG GCCTTGGTATGTTCCTGCTTCATCCCTTTCTACAGTGGCCTTATCCCTCCTTCCTTCAGAGGCGTG GTAAGTCGGCTTTCTCTGCTAGCGCTGAGTCCTGGGGGCCTCTGAAGTGTGCTCACACATCTCCTG CCTGCAGGGCACTGGTGTCGGGCACCTCAGGGTCTGTCCCATGGTGGAGCCCCATGCCTCACTGCC TTTCAGACAGAGTAGCCACAGCTGGCCCTATTTCCAGGCTACCCGGGCAGCAAAACTTACTGCATG TGTAATTAATTATTTGGCTATCTGTAAGGTAAACTGGCTGGTTCACTTAATCTGCACCTTAAGCAT CAGATAGCTTCTCAGTGATCTAGTTAAACTATATGATGTTGGCCAGGCGCGGTGGCTCATGTCTGT AATCCCAGCACTTTGGGAGCCTGAAGCAGGCAGATCACTTGAGGTCAGGAGTTCGAGACCAGCCTG GCCAACAGTGTGAAACTCTGTCTCTCCTAAAAATACAAAAATTAGCTGGGCATGGTGGTGTGCACC TGTAATCCCAGCTGCTCGGGAGGCTGAGGCAGGAGAATTGCTTGAACTTGGGA SEQ PNPLA3 TGCCTGCTGACTGCTCTGTAGCACAGTGCTTCG IDrs738409 CAAAGTGTGATCCTGGGACCAGCAGAGCAGCAG NO:CTCCTTTGAGCTTATTGGAATGGCAGACCCTCA 414 GGTCCCACCTCTGACCTGCTGCATGGGAATTCTGGGGAGGGACGCAGAATCTCTGGTTCCACAGGC TCTCCGGTGATGCTAATGAATACCGGCATTTGAACAGCACCGATCTAGCCCCTTTCAGTCCATGAG CCAACAACCCTTGGTCCTGTCTGTGGTGACCCAGTGTGACTCTCATGGGGAGCAAGGAGAGGAAGT TGAAGTTCACTGACAGGGTTGTTAAGGGGATTATGCAATAGATGAGACCCATGGGCCTGAAGTCCG AGGGTGTATGTTAGTTCCCCGTTCTTTTGACCCATGGATTAACCTACTCTGTGCAAAGGGCATTTT CAAGTTTGTTGCCCTGCTCACTTGGAGAAAGCTTATGAAGGATCAGGAAAATTAAAAGGGTGCTCT CGCCTATAACTTCTCTCTCCTTTGCTTTCACAGGCCTTGGTATGTTCCTGCTTCATGCCCTTCTAC AGTGGCCTTATCCCTCCTTCCTTCAGAGGCGTGGTAAGTCGGCTTTCTCTGCTAGCGCTGAGTCCT GGGGGCCTCTGAAGTGTGCTCACACATCTCCTGCCTGCAGGGCACTGGTGTCGGGCACCTCAGGGT CTGTCCCATGGTGGAGCCCCATGCCTCACTGCCTTTCAGACAGAGTAGCCACAGCTGGCCCTATTT CCAGGCTACCCGGGCAGCAAAACTTACTGCATGTGTAATTAATTATTTGGCTATCTGTAAGGTAAA CTGGCTGGTTCACTTAATCTGCACCTTAAGCATCAGATAGCTTCTCAGTGATCTAGTTAAACTATA TGATGTTGGCCAGGCGCGGTGGCTCATGTCTGTAATCCCAGCACTTTGGGAGCCTGAAGCAGGCAG ATCACTTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACAGTGTGAAACTCTGTCTCTCCTAAAAA TACAAAAATTAGCTGGGCATGGTGGTGTGCACCTGTAATCCCAGCTGCTCGGGAGGCTGAGGCAGG AGAATTGCTTGAACTTGGGA SEQ PNPLA3 WTTGCCTGCTGACTGCTCTGTAGCACAGTGCTTCG ID CAAAGTGTGATCCTGGGACCAGCAGAGCAGCAGNO: CTCCTTTGAGCTTATTGGAATGGCAGACCCTCA 415GGTCCCACCTCTGACCTGCTGCATGGGAATTCT GGGGAGGGACGCAGAATCTCTGGTTCCACAGGCTCTCCGGTGATGCTAATGAATACCGGCATTTGA ACAGCACCGATCTAGCCCCTTTCAGTCCATGAGCCAACAACCCTTGGTCCTGTCTGTGGTGACCCA GTGTGACTCTCATGGGGAGCAAGGAGAGGAAGTTGAAGTTCACTGACAGGGTTGTTAAGGGGATTA TGCAATAGATGAGACCCATGGGCCTGAAGTCCGAGGGTGTATGTTAGTTCCCCGTTCTTTTGACCC ATGGATTAACCTACTCTGTGCAAAGGGCATTTTCAAGTTTGTTGCCCTGCTCACTTGGAGAAAGCT TATGAAGGATCAGGAAAATTAAAAGGGTGCTCTCGCCTATAACTTCTCTCTCCTTTGCTTTCACAG GCCTTGGTATGTTCCTGCTTCATCCCCTTCTACAGTGGCCTTATCCCTCCTTCCTTCAGAGGCGTG GTAAGTCGGCTTTCTCTGCTAGCGCTGAGTCCTGGGGGCCTCTGAAGTGTGCTCACACATCTCCTG CCTGCAGGGCACTGGTGTCGGGCACCTCAGGGTCTGTCCCATGGTGGAGCCCCATGCCTCACTGCC TTTCAGACAGAGTAGCCACAGCTGGCCCTATTTCCAGGCTACCCGGGCAGCAAAACTTACTGCATG TGTAATTAATTATTTGGCTATCTGTAAGGTAAACTGGCTGGTTCACTTAATCTGCACCTTAAGCAT CAGATAGCTTCTCAGTGATCTAGTTAAACTATATGATGTTGGCCAGGCGCGGTGGCTCATGTCTGT AATCCCAGCACTTTGGGAGCCTGAAGCAGGCAGATCACTTGAGGTCAGGAGTTCGAGACCAGCCTG GCCAACAGTGTGAAACTCTGTCTCTCCTAAAAATACAAAAATTAGCTGGGCATGGTGGTGTGCACC TGTAATCCCAGCTGCTCGGGAGGCTGAGGCAGGAGAATTGCTTGAACTTGGGA

Guide RNAs were designed to detect the wild type sequence or thesequence with a mutation at the second SNP (“rs738409”), but ignore thesequence with the mutation at the first SNP (“rs738408”). Guide RNAscorresponding to SEQ ID NO: 300-SEQ ID NO: 319 were directed to the wildtype (SEQ ID NO: 415, “WT”) sequence on the forward strand at position1-position 20, respectively. gRNAs corresponding to SEQ ID NO: 320-SEQID NO: 339 were directed to the wild type (“WT”) sequence on the reversestrand at position 1-position 20, respectively. gRNAs corresponding toSEQ ID NO: 340-SEQ ID NO: 359 were directed to the mutant (SEQ ID NO:414, “rs738409”) sequence on the forward strand at position 1-position20, respectively. gRNAs corresponding to SEQ ID NO: 360-SEQ ID NO: 379were directed to the mutant (“rs738409”) sequence on the reverse strandat position 1-position 20, respectively. Each gRNA was used to detectfour different target sequences corresponding to the wild type sequence(SEQ ID NO: 415, “WT”), a sequence with a point mutation at a first site(SEQ ID NO: 413, “rs738408”), a sequence with a point mutation at asecond site (SEQ ID NO: 414, “rs738409”), or a sequence with pointmutations at both the first site and the second site (SEQ ID NO: 412,“rs738409+rs738408”).

Detection reactions were carried out using gRNAs designed to targetdifferent positions on the target nucleic acid relative to the positionof the SNP on either the forward or reverse strand (shown on the x-axisis the position relative to the SNP on either the forward or reversestrand). Upon hybridization of gRNA to the target nucleic acid, SEQ IDNO: 11 is activated and trans cleaves a reporter (SEQ ID NO: 119 withN-terminal /56-FAM/ and C-terminal /3IABkFQ/), releasing a fluorescentdetectable label. Detection of target nucleic acids with SNPs wascarried out by measuring fluorescence from the cleaved detectable label,and the maximum rate (“Max Rate (AU/min)”) was calculated asfluorescence units per minute. gRNAs that exhibited specificity for thewild type sequence (“WT”) or the sequence with the mutation at thesecond SNP (“rs738409”), but did not non-specifically detect thesequence with the mutation at the first SNP (“rs738408”), are indicatedby black arrows. Sequences of the gRNAs used are provided in TABLE 26.

TABLE 26 gRNAs for Detection of Target Sequences with ArtificallyIntroduced PAMs SEQ ID NO: Target gRNA Sequence SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUCCCCUUCUACAGUGGCCUUA 300 1 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUUCCCCUUCUACAGUGGCCUU 301 2 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUAUCCCCUUCUACAGUGGCCU 302 3 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUCAUCCCCUUCUACAGUGGCC 303 4 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUUCAUCCCCUUCUACAGUGGC 304 5 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUUUCAUCCCCUUCUACAGUGG 305 6 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUCUUCAUCCCCUUCUACAGUG 306 7 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUGCUUCAUCCCCUUCUACAGU 307 8 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUUGCUUCAUCCCCUUCUACAG 308 9 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUCUGCUUCAUCCCCUUCUACA 309 10 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUCCUGCUUCAUCCCCUUCUAC 310 11 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUUCCUGCUUCAUCCCCUUCUA 311 12 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUUUCCUGCUUCAUCCCCUUCU 312 13 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUGUUCCUGCUUCAUCCCCUUC 313 14 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUUGUUCCUGCUUCAUCCCCUU 314 15 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUAUGUUCCUGCUUCAUCCCCU 315 16 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUUAUGUUCCUGCUUCAUCCCC 316 17 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUGUAUGUUCCUGCUUCAUCCC 317 18 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUGGUAUGUUCCUGCUUCAUCC 318 19 SEQ ID NO: WT-FWD-UAAUUUCUACUAAGUGUAGAUUGGUAUGUUCCUGCUUCAUC 319 20 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUGAUGAAGCAGGAACAUACCA 320 1 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUGGAUGAAGCAGGAACAUACC 321 2 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUGGGAUGAAGCAGGAACAUAC 322 3 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUGGGGAUGAAGCAGGAACAUA 323 4 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUAGGGGAUGAAGCAGGAACAU 324 5 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUAAGGGGAUGAAGCAGGAACA 325 6 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUGAAGGGGAUGAAGCAGGAAC 326 7 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUAGAAGGGGAUGAAGCAGGAA 327 8 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUUAGAAGGGGAUGAAGCAGGA 328 9 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUGUAGAAGGGGAUGAAGCAGG 329 10 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUUGUAGAAGGGGAUGAAGCAG 330 11 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUCUGUAGAAGGGGAUGAAGCA 331 12 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUACUGUAGAAGGGGAUGAAGC 332 13 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUCACUGUAGAAGGGGAUGAAG 333 14 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUCCACUGUAGAAGGGGAUGAA 334 15 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUGCCACUGUAGAAGGGGAUGA 335 16 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUGGCCACUGUAGAAGGGGAUG 336 17 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUAGGCCACUGUAGAAGGGGAU 337 18 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUAAGGCCACUGUAGAAGGGGA 338 19 SEQ ID NO: WT-REV-UAAUUUCUACUAAGUGUAGAUUAAGGCCACUGUAGAAGGGG 339 20 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUGCCCUUCUACAGUGGCCUUA 340 FWD-1 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUUGCCCUUCUACAGUGGCCUU 341 FWD-2 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUAUGCCCUUCUACAGUGGCCU 342 FWD-3 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUCAUGCCCUUCUACAGUGGCC 343 FWD-4 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUUCAUGCCCUUCUACAGUGGC 344 FWD-5 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUUUCAUGCCCUUCUACAGUGG 345 FWD-6 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUCUUCAUGCCCUUCUACAGUG 346 FWD-7 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUGCUUCAUGCCCUUCUACAGU 347 FWD-8 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUUGCUUCAUGCCCUUCUACAG 348 FWD-9 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUCUGCUUCAUGCCCUUCUACA 349 FWD-10 SEQ ID NO:rs738409- UAAUUUCUACUAAGUGUAGAUCCUGCUUCAUGCCCUUCUAC 350 FWD-11SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUUCCUGCUUCAUGCCCUUCUA 351FWD-12 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUUUCCUGCUUCAUGCCCUUCU352 FWD-13 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUGUUCCUGCUUCAUGCCCUUC 353 FWD-14 SEQ ID NO:rs738409- UAAUUUCUACUAAGUGUAGAUUGUUCCUGCUUCAUGCCCUU 354 FWD-15SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUAUGUUCCUGCUUCAUGCCCU 355FWD-16 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUUAUGUUCCUGCUUCAUGCCC356 FWD-17 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUGUAUGUUCCUGCUUCAUGCC 357 FWD-18 SEQ ID NO:rs738409- UAAUUUCUACUAAGUGUAGAUGGUAUGUUCCUGCUUCAUGC 358 FWD-19SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUUGGUAUGUUCCUGCUUCAUG 359FWD-20 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUCAUGAAGCAGGAACAUACCA360 REV-1 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUGCAUGAAGCAGGAACAUACC361 REV-2 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUGGCAUGAAGCAGGAACAUAC362 REV-3 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUGGGCAUGAAGCAGGAACAUA363 REV-4 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUAGGGCAUGAAGCAGGAACAU364 REV-5 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUAAGGGCAUGAAGCAGGAACA365 REV-6 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUGAAGGGCAUGAAGCAGGAAC366 REV-7 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUAGAAGGGCAUGAAGCAGGAA367 REV-8 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUUAGAAGGGCAUGAAGCAGGA368 REV-9 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUGUAGAAGGGCAUGAAGCAGG369 REV-10 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUUGUAGAAGGGCAUGAAGCAG 370 REV-11 SEQ ID NO:rs738409- UAAUUUCUACUAAGUGUAGAUCUGUAGAAGGGCAUGAAGCA 371 REV-12SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUACUGUAGAAGGGCAUGAAGC 372REV-13 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUCACUGUAGAAGGGCAUGAAG373 REV-14 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUCCACUGUAGAAGGGCAUGAA 374 REV-15 SEQ ID NO:rs738409- UAAUUUCUACUAAGUGUAGAUGCCACUGUAGAAGGGCAUGA 375 REV-16SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUGGCCACUGUAGAAGGGCAUG 376REV-17 SEQ ID NO: rs738409- UAAUUUCUACUAAGUGUAGAUAGGCCACUGUAGAAGGGCAU377 REV-18 SEQ ID NO: rs738409-UAAUUUCUACUAAGUGUAGAUAAGGCCACUGUAGAAGGGCA 378 REV-19 SEQ ID NO:rs738409- UAAUUUCUACUAAGUGUAGAUUAAGGCCACUGUAGAAGGGC 379 REV-20

In a third assay, sensitivity of the detection assay to single- anddouble-point mutations was tested. As used in the assay shown in FIG.78, the PNPLA3 target sequence with two SNP sites was capable of beingdetected by certain gRNAs. Sensitivity of a programmable nuclease of SEQID NO: 11 to SNP mutations was tested individually and in combination.FIG. 78 shows detection of single and double mutations in a targetnucleic acid sequence using a programmable nuclease of SEQ ID NO: 11.Target sequences corresponding to SEQ ID NO: 412-SEQ ID NO: 415 weredetected. Samples containing a target nucleic acid (“target”) witheither a wild type nucleic acid sequence (SEQ ID NO: 415, “WT”), asequence with a mutation at a first SNP (SEQ ID NO: 413, “rs738408”), asequence with a mutation at a second SNP (SEQ ID NO: 414, “rs738409”),or a sequence with mutations at the first SNP and the second SNP (SEQ IDNO: 412, “rs738409/408”) were detected using a gRNA directed to the wildtype sequence (SEQ ID NO: 301, “R1287-WT”), a gRNA directed to thesequence with a mutation at a second SNP (SEQ ID NO: 341,“R1327-rs738409”), a gRNA directed to the sequence with a mutation at afirst SNP (SEQ ID NO: 421, “R1434-rs738408”), or a gRNA directed to thesequence with mutations at the first SNP and the second SNP (SEQ ID NO:422, “R1435-rs738409/408”). Detection was measured using fluorescence,and the maximum rate (“Max Rate (AU/min)”) was calculated asfluorescence units per minute. The results showed that gRNAs could bedesigned that were specific to each of the wild type sequence (SEQ IDNO: 415, “WT”), the sequence with a mutation at a first SNP (SEQ ID NO:413, “rs738408”), the sequence with a mutation at a second SNP (SEQ IDNO: 414, “rs738409”), or the sequence with mutations at both the firstSNP and the second SNP (SEQ ID NO: 412, “rs738409/408”). Furthermore, aprogrammable nuclease of SEQ ID NO: 11 was sensitive to single anddouble mutations and gRNAs were designed to detect all allelepossibilities. NTC shows control experiments without the target nucleicacid.

In a fourth assay, the functionality of gRNAs targeted to specific pointmutations in combined pools was tested. The wild type, single-, anddouble-point mutants tested in the assay shown in FIG. 78 were testedwith pooled gRNAs. FIG. 79 shows detection of two PNPLA3 SNPs in atarget nucleic acid sequence without a native PAM using a programmablenuclease of SEQ ID NO: 11. Target nucleic acids containing the wild typesequence (SEQ ID NO: 415, “WT”), a sequence with a mutation at a firstSNP (SEQ ID NO: 413, “rs738408”), a sequence with a mutation at a secondSNP (SEQ ID NO: 414, “rs738409”), or a sequence with mutations at thefirst SNP allele and the second SNP (SEQ ID NO: 412, “rs738409/408”). Asample without a target sequence (non-target control, “NTC”) was used asa negative control. Guide RNAs designed to detect the wild type sequenceor the sequence with a mutation at the first SNP (SEQ ID NO: 301 and SEQID NO: 421, “WT DETECTR”) were pooled to detect the target nucleic acidin each sample type. Guide RNAs designed to detect the sequence with amutation at the second SNP (SEQ ID NO: 341 and SEQ ID NO: 422, “rs738409DETECTR”) were pooled to detect the target nucleic acid in each sampletype. Detection was measured using fluorescence, and the maximum rate(“Max Rate (AU/min)”) was calculated as fluorescence units per minute.The results showed that gRNAs could be pooled to selectively detect SNPsof interest and not other nearby genetic variation. A first gRNA poolwas able to detect both the wild type sequence (SEQ ID NO: 415, “WT”)and the sequence with a mutation at a first SNP (SEQ ID NO: 413,“rs738408”), while a second gRNA pool was able to detect the sequencewith a mutation at a second SNP (SEQ ID NO: 414, “rs738409”) and thesequence with mutations at both the first SNP and the second SNP (SEQ IDNO: 412, “rs738409/408”). Guide RNA sequences used in each pool areprovided in TABLE 27.

TABLE 27 gRNAs Pools for Detection of two PNPLA3 SNP Alleles SEQ ID gRNANO: Pool gRNA Sequence SEQ ID WT  UAAUUUCUACUAAGUGUAGAUUCCCCUU NO: 301DETECTR CUACAGUGGCCUU gRNAs SEQ ID WT  UAAUUUCUACUAAGUGUAGAUUCCCUUUNO: 421 DETECTR CUACAGUGGCCUU gRNAs SEQ ID Rs738409UAAUUUCUACUAAGUGUAGAUUGCCCUU NO: 341 DETECTR CUACAGUGGCCUU gRNAs SEQ IDRs738409 UAAUUUCUACUAAGUGUAGAUUGCCUUU NO: 422 DETECTR CUACAGUGGCCUUgRNAs

Example 28 LbCas12a SNP Sensitivity Along a Target Sequence

This example describes sensitivity of LbCas12a to SNPs positioned alonga target sequence. In a first assay, sensitivity to point mutations in atarget sequence with a native PAM site was tested. To determine whichpositions along a target nucleic acid sequence were most sensitive tosingle point mutations, all four nucleotide possibilities (A, T, C, orG) at each position were tiled along a target of a target nucleic acidsequence. The assay was performed for two target nucleic acid sequences,a HERC2 target nucleic acid sequence and an ALDH target nucleic acidsequence. Both target nucleic acid sequences comprised a native PAMsite. The target nucleic acids comprising the PAM site with each of allpossible point mutations were detected with using LbCas12a (SEQ ID NO:1).

FIG. 80 shows detection of single point mutations at different positionsalong a nucleic acid sequence using LbCas12a (SEQ ID NO: 1). Pointmutations to each nucleic acid (A, T, C, or G, “SNP Base (target)”) weremade along a target nucleic acid sequence at different positionsrelative to a native PAM. To determine the sensitivity of the detectionassay to single point mutations (e.g., a SNP), the target nucleic acidwas detected using a gRNA directed to hybridize to the wild typesequence. Black circles label with “WT” indicate nucleotide at eachposition of the wild type sequence that is reverse complementary to thegRNA sequence. The assay was performed with a HERC2 target sequence (toppanel, wild type sequence corresponding to SEQ ID NO: 416) or an ALDHtarget sequence (bottom panel, wild type sequence corresponding to SEQID NO: 417). Upon hybridization of gRNA to the target nucleic acid, SEQID NO: 11 is activated and trans cleaves a reporter, releasing afluorescent detectable label. The HERC2 sequence was detected using agRNA corresponding to SEQ ID NO: 246 (top plot) and the ALDH sequencewas detected using a gRNA corresponding to SEQ ID NO: 425 (bottom plot).Detection of target nucleic acids with SNPs was carried out by measuringfluorescence from the cleaved detectable label, and the maximum rate(“Average Max Rate”) was calculated as fluorescence units per minute andaveraged between four replicates. Results indicated that LbCas12a wassensitive to point mutations along the entire length of the gRNA targetsite. The specificity for individual point mutations depended onsequence context of the target nucleic acid.

In a second assay, LbCas12a (SEQ ID NO: 1) was used to detect variantsat two SNP sites in a target nucleic acid sequence without a native PAM.The detection assay was run for 90 minutes at 37° C. with either a wildtype DNA (“WT”), a target DNA with a mutation at a first SNP(“rs738408”), or a target DNA with a mutation at a second SNP(“rs738409”).

FIG. 81 shows detection of two PNPLA3 SNPs in a target nucleic acidsequence without a native “TTTN” PAM sequence using LbCas12a (SEQ ID NO:1). Target nucleic acids tested contained the wild type sequence (SEQ IDNO: 415, “WT”), a sequence with a mutation at a first SNP (SEQ ID NO:413, “rs738408”), a sequence with a mutation at a second SNP (SEQ ID NO:414, “rs738409”), or a sequence with mutations at the first SNP alleleand the second SNP (SEQ ID NO: 412, “rs738409/408”). Guide RNAs weredesigned to detect the wild type sequence (“WT specific”) or thesequence with a mutation at the second SNP (“rs738409 specific”). GuideRNAs corresponding to SEQ ID NO: 300-SEQ ID NO: 319 were directed to thewild type (SEQ ID NO: 415, “WT”) sequence on the forward strand atposition 1-position 20, respectively. gRNAs corresponding to SEQ ID NO:320-SEQ ID NO: 339 were directed to the wild type (“WT”) sequence on thereverse strand at position 1-position 20, respectively. gRNAscorresponding to SEQ ID NO: 340-SEQ ID NO: 359 were directed to themutant (SEQ ID NO: 414, “rs738409”) sequence on the forward strand atposition 1-position 20, respectively. gRNAs corresponding to SEQ ID NO:360-SEQ ID NO: 379 were directed to the mutant (“rs738409”) sequence onthe reverse strand at position 1-position 20, respectively. Each gRNAwas used to detect four different target sequences corresponding to thewild type sequence (SEQ ID NO: 415, “WT”), a sequence with a pointmutation at a first site (SEQ ID NO: 413, “rs738408”), a sequence with apoint mutation at a second site (SEQ ID NO: 414, “rs738409”), or asequence with point mutations at both the first site and the second site(SEQ ID NO: 412, “rs738409+rs738408”). Sequences of the gRNAs used inthis experiment are provided in TABLE 26.

Detection reactions were carried out using gRNAs designed to targetdifferent positions on the target nucleic acid relative to the positionof the SNP on either the forward or reverse strand (shown on the x-axisis the position relative to the SNP on either the forward or reversestrand). Upon hybridization of gRNA to the target nucleic acid, LbCas12awas activated and trans cleaved a reporter (SEQ ID NO: 119 withN-terminal /56-FAM/ and C-terminal /3IABkFQ/), releasing a fluorescentdetectable label. Detection of target nucleic acids with SNPs wascarried out by measuring fluorescence from the cleaved detectable label,and the maximum rate (“Max Rate (AU/min)”) was calculated asfluorescence units per minute. gRNAs that exhibited specificity for thewild type sequence or the sequence with the mutation at the second SNPare indicated by arrows. The results demonstrated that certain gRNAswere specific for the wild type sequence (“WT”) or the sequence with themutation at the second SNP (“rs738409”), but did not non-specificallydetect the sequence with the mutation at the first SNP (“rs738408”).

Example 29 CasY3 SNP Sensitivity Along a CYP2C19*2 SNP Target Sequence

This example describes sensitivity of CasY3 to the CYP2C19*2 SNPpositioned along a target sequence. The ability for CasY3 todiscriminate single point mutations is tested. The SNP sensitivity ofCasY3 for the CYP2C19*2 SNP on sequences with and without native TR PAMsis tested. Target nucleic acids having either a wild type sequence(“WT”) or a sequence with a single point mutation (“mt”) are detectedusing CasY3. Target DNA is detected at a concentration of 1 nM in thedetection assay, which is run for 90 minutes at 37° C. Target nucleicacid sequences with or without a native TR PAM using CasY3 (SEQ ID NO:282) are detected. Samples comprise a wild type target nucleic acidsequence or a sequence with a mutation at a SNP are detected with gRNAscomprising a crRNA and a scout RNA (sctRNA) designed to target differentpositions relative on the target nucleic acid relative to the positionof the SNP on either the forward strand or the reverse strand.

crRNAs are directed to the wild type sequence on the forward strand atposition 1-position 18 on a target nucleic acid, respectively. crRNAscorresponding to are directed to the wild type sequence on the reversestrand at position 1-position 18 on a target nucleic acid, respectively.Upon hybridization of gRNA to the target nucleic acid, CasY3 isactivated and trans cleaves a reporter, releasing a fluorescentdetectable label. Detection of target nucleic acids with SNPs is carriedout by measuring fluorescence from the cleaved detectable label. Themaximum rate (“Max Rate (AU/min)”) is calculated as fluorescence unitsper minute. gRNAs are identified that are specific for the wild typesequence or the sequence with the mutation at the second SNP.

Example 30 LbuCas13a SNP Sensitivity Along a Target Sequence

This example describes sensitivity of LbuCas13a to SNPs positioned alonga target sequence. RNA and ssDNA target nucleic acid sequences weretested. In a first assay, sensitivity to point mutations in an RNAtarget nucleic acid was tested. To determine which positions along anRNA target nucleic acid sequence were most sensitive to single pointmutations, all four nucleotide possibilities (A, T, C, or G) at eachposition were tiled along a target of a target nucleic acid sequence.The assay was performed for a target RNA sequence from influenza Avirus. The RNA target nucleic acid with each of all possible pointmutations was detected with using LbuCas13a (SEQ ID NO: 104). The RNAtarget nucleic acid was detected at 0.25 nM target, and the assay wasrun for 20 minutes.

FIG. 82 shows detection of single point mutations at different positionsalong target RNA sequence (SEQ ID NO: 748) using LbuCas13a (SEQ ID NO:104). Point mutations to each nucleic acid (A, T, C, or G, “SNP Base(target)”) were made along a target nucleic acid sequence at differentpositions relative to a native PAM. To determine the sensitivity of thedetection assay to single point mutations (e.g., a SNP), the targetnucleic acid was detected using a gRNA directed to hybridize to the wildtype sequence. Black circles label with “WT” indicate nucleotide at eachposition of the wild type sequence that is reverse complementary to thegRNA sequence. Data is not shown for wild type positions (black circleslabeled with “WT”). Detection of the wild type sequence is shown in thesquare marked “WT” at SNP position 1. Detection of a negative control(water) is shown in the square marked “None” at position “None.” Thetargets were detected using a gRNA corresponding to SEQ ID NO: 507. Uponhybridization of gRNA to the target nucleic acid, LbuCas13a wasactivated and trans cleaved a reporter, releasing a fluorescentdetectable label. Detection of target nucleic acids with SNPs wascarried out by measuring fluorescence from the cleaved detectable label.Results showed that LbuCas13a was able to differentiate certain singlepoint mutations at some positions along the RNA target sequence. Sitesat which LbuCas13a was able to distinguish all four nucleotide positionsare indicated by arrows.

In a second assay, sensitivity to point mutations in a ssDNA targetnucleic acid was tested. To determine which positions along a ssDNAtarget nucleic acid sequence were most sensitive to single pointmutations, all four nucleotide possibilities (A, T, C, or G) were tiledalong a target of a target nucleic acid sequence. The assay wasperformed for a target ssDNA sequence from influenza A virus. The RNAtarget nucleic acid with each of all possible point mutations wasdetected with using LbuCas13a (SEQ ID NO: 104). The RNA target nucleicacid was detected at 2.5 nM target, and the assay was run for 20 minutesat 37° C. FIG. 83 shows detection of single point mutations at differentpositions along target ssDNA (SEQ ID NO: 749) sequence using LbuCas13a(SEQ ID NO: 104). Point mutations to each nucleic acid (A, T, C, or G,“SNP Base (target)”) were made along a target nucleic acid sequence witha wild type sequence corresponding to SEQ ID NO: 407(TCTACGCTGCAGTCCTCGCT) at different positions relative to a native PAM.To determine the sensitivity of the detection assay to single pointmutations (e.g., a SNP), the target nucleic acid was detected using agRNA directed to the wild type sequence. Black circles label with “WT”indicate the nucleotide at each position of the wild type sequence thatis reverse complementary to the gRNA sequence. Data is not shown forwild type positions (black circles labeled with “WT”). Detection of thewild type sequence is shown in the square marked “WT” at SNP position 1.Detection of a negative control (water) is shown in the square marked“None” at position “None.” The targets were detected using a gRNAcorresponding to SEQ ID NO: 507. Upon hybridization of gRNAs to thetarget nucleic acid, LbuCas31a was activated and trans cleaved areporter, releasing a fluorescent detectable label. Detection of targetnucleic acids with SNPs was carried out by measuring fluorescence fromthe cleaved detectable label. Results showed that LbuCas13a was able todifferentiate certain single point mutations at some positions along thessDNA target sequence. Sites at which LbuCas13a was able to distinguishall four nucleotide mutations are indicated by black arrows. Sites atwhich LbuCas13a was able to distinguish at least one nucleotide mutationare indicated by gray arrows.

Example 31 Detection of a Nucleic Acid Amplified with dUTPs UsingDETECTR

This example describes detection of a nucleic acid that had beenamplified with dUTPs using a DETECTR reaction. Two target nucleic acidsequences, ALDH2 wild type sequence and ALDH2 with a single pointmutation at T790M (“T790M”), were amplified in a PCR reaction performedwith dUTP nucleotide bases in place of dTTP nucleotide bases. Each PCRamplification reaction included 1 μM forward primer, 1 μM reverseprimer, Taq polymerase (in Taq master mix), UDG enzyme, and a templatesequence with either the ALDH2 wild type sequence or ALDH2 T790M mutantsequence. The UDG enzyme was heat-activated at 50° C. prior toamplification to degrade nucleic acid contaminants containing dU bases.The UDG enzyme was subsequently inactivated at 90° C. before initiatingPCR amplification of the target nucleic acid. Amplification of thetarget sequence was verified by gel electrophoresis. The ALDH2 wild typesequence was successfully amplified. In an alternate assayconfiguration, a thermolabile UDG enzyme was used in place of the UDGenzyme. The thermolabile UDG enzyme was activated at 25° C. for 10minutes prior to amplification to degrade nucleic acid contaminantscontaining dU bases. The thermolabile UDG enzyme was subsequentlyinactivated at a temperature of at least 50° C. before initiating PCRamplification of the target nucleic acid.

The amplified ALDH2 wild type sequence with dUTPs was detected using anLbCas12a detection assay. The ALDH2 wild type sequence was detected witheither a gRNA directed to hybridize to the wild type sequence (“ALDH2(WT SNP”)) or a gRNA directed to hybridize to the T790M mutant sequence(“ALDH2 (Mutant SNP)”). Each detection reaction included 1.25 μM gRNA,200 nM LbCas12a (SEQ ID NO: 1), 100 nM ssDNA-FQ reporter substrate, andthe target sequence containing dUTPs.

FIG. 71A-FIG. 71B show the detection of ALDH2 WT allele from humangenomic DNA (SEQ ID NO: 417) with dU-containing amplicons with Cas12.The ALDH2 gene was amplified from human saliva containing the WT alleleusing Taq master mix containing dUTP in place of dTTP, such that all Tnucleotides with the annotated ALDH2 target sequence shown in FIG. 71Ahave been replaced by U nucleotides. The amplicon was added directly toa Cas12 DETECTR assay. Cas12 guide RNAs targeting the ALDH2 WT alleledetected only the cognate WT sequence and not the mutant allele,demonstrating that Cas12 is capable of SNP detection with dU-containingtargets. FIG. 71B shows a DETECTR reaction of an ALDH2 target nucleicacid sequence amplified with dUTPs using LbCas12a (SEQ ID NO: 1).Fluorescence was measured over time in the presence of the wild typenucleic acid sequence (“WT SNP”, top most line), a sequence with a pointmutation (“Mutant SNP”, middle line), or a negative control without thetarget nucleic acid sequence (bottom line). Samples were detected withgRNAs directed to hybridize to the wild type sequence. Results showedthat the LbCas12a detection assay detected target nucleic acid sequencesamplified with dUTPs. The detection assay was specific for the gRNAdirected to the wild type sequence and did not non-specifically detectthe wild type sequence with the gRNA directed to hybridize to the T790Mmutant sequence. Primers used in this assay are provided in TABLE 22.

To verify that the amplified product detected in the LbCas12a detectionreaction contained dUTPs, the target nucleic acid was contacted with UDGenzyme which degrades nucleic acid sequences with dUTPs. The amplifiedtarget sequence was successfully degraded by UDG. Degradation of thetarget sequence by UDG was verified by gel electrophoresis.

Example 32 In Vitro Transcription of a Nucleic Acid Reverse Transcribedand Amplified with dUTPs

This example describes in vitro transcription of a nucleic acid that hadbeen reverse transcribed and amplified with dUTPs. Target RNA nucleicacid sequences were amplified using primers directed to sites with theprostate cancer biomarkers PCA3, PSA, and T2ERG. The target RNAsequences were reverse transcribed using a reverse transcriptase enzyme.The reverse transcribed DNA was then amplified using PCR with dUTPnucleic acid bases in place of dTTP nucleic acid bases. Amplificationwas verified by gel electrophoresis. The amplified DNA was thentranscribed into RNA using an in vitro transcription reaction with a T7RNA polymerase.

Example 33 Detection of Chlamydia Using PCR, IVT, and DETECTR

This example describes detection of chlamydia nucleic acids in a sampleusing PCR, in vitro transcription (IVT) and DETECTR. Detectionsensitivity for chlamydia nucleic acids was improved by amplifying achlamydia target nucleic acid sequence and reverse transcribing theamplified sequence by in vitro transcription. The target sequence wasPCR amplified using dUTPs in place of dTTPs, as described in EXAMPLE 31.The amplified PCR product was used as a template for the in vitrotranscription reaction. In vitro transcription reaction was performedwith a T7 RNA polymerase. The amplified and transcribed nucleic acidsequence was detected using an LbuCas13a detection assay. The detectionassay was performed with 40 nM gRNA and 40 nM LbuCas13a (SEQ ID NO:104). The reaction was run for 30 minutes at 37° C. Followingincubation, FAM-U5 reporter and RNase inhibitor were added to thereaction mixture and fluorescence was measured.

FIG. 84 shows detection of a Chlamydia trachomatis target nucleic acidsequence with LbuCas13a (SEQ ID NO: 104) following polymerase chainreaction (PCR) amplification and in vitro transcription (IVT) of samplesthat were either positive or negative for Chlamydia. Targets weredetected with either a gRNA targeted to Chlamydia 5S rRNA (SEQ ID NO:418), a gRNA targeted to Chlamydia 16S rRNA (SEQ ID NO: 419), or anoff-target gRNA (SEQ ID NO: 420). Fluorescence was measured over time.An increase in fluorescence indicated detection of the target nucleicacid sequence. Thirty-one samples either positive or negative forChlamydia or a negative control with no target nucleic acid weredetected. Fluorescence over time detected in each sample is shown inindividual plots. The top left plot shows the negative control. Eachsample was detected with each of three different gRNAs (SEQ ID NO: 418,“CT001-33;” SEQ ID NO: 419, “SSU-1368;” or SEQ ID NO: 410, “C”), asshown by individual traces in each plot. Sequences for the gRNAs areprovided in TABLE 28.

TABLE 28 gRNAs for Detection of a Chlamhydia Target Nucleic Acid SEQ IDgRNA NO: Name gRNA Sequence SEQ ID CT001-33GGCCACCCCAAAAAUGAAGGGGACUAAAAC NO: 418 ACUUCUGAGUUCGGAAUGGUG SEQ IDSSU-1368 GGCCACCCCAAAAAUGAAGGGGACUAAAAC NO: 419 AACGUAUUCACGGCGUUAUGGSEQ ID R003 GGCCACCCCAAAAAUGAAGGGGACUAAAAC NO: 420 AGUGAUAAGUGGAAUGCCAUG

FIG. 85 shows heatmaps of the fluorescence detected in FIG. 84 (right).Panels on the right indicate the maximum fluorescent rate detected witheither a gRNA targeting a Chlamydia 16S RNA sequence (SEQ ID NO: 419,“16S gRNA”), a gRNA targeting a Chlamydia 5S RNA sequence (SEQ ID NO:418, “5S gRNA”), or a gRNA not directed to a Chlamydia target sequence(SEQ ID NO: 420, “off-target gRNA”). Shaded boxes in the left column(“Ct”) indicate that the sample was positive for Chlamydia. Resultsshowed that SSU-1368 (SEQ ID NO: 419) is more specific for the chlamydiatarget sequence than CT001-33 (SEQ ID NO: 418).

Example 34 Identification of Cas12 Variants with Trans Cleavage Activity

This example describes the identification of Cas12 variants with transcleavage activity. Different Cas12 variants corresponding to SEQ ID NO:11, SEQ ID NO: 3, and SEQ ID NO: 571-SEQ ID NO: 589 were tested fortrans cleavage activity as well as sensitivity and specificity fortarget sequences.

In a first assay, each Cas12 variant was tested for sensitivity andability to detect a target nucleic acid sequence with different PAMsequences. DETECTR trans cleavage assays were performed in the presenceof activity buffer (5 mM MgCl₂, 20 mM, Tris pH 7.5, 120 mM NaCl, and 1%glycerol). Different dsDNA target nucleic acids with different PAMsequences were detected at a final concentration of 100 nM. ssDNAreporters were present in each reaction at a concentration of 50 nM.Target dsDNA was obtained by annealing complementary ssDNA primers at aratio of 2:1 of non-target strand to target strand in hybridizationbuffer (50 mM NaCl, 1 mM Tris pH 8.0, 0.1 mM EDTA), to ensuredouble-stranded DNA is being detected instead of single-stranded DNA.Pre-crRNA was ordered from Synthego. The protein of interest and theguide RNA were added to each tube and incubated for 20 minutes at 37° C.Each reaction contained 16 μL of the incubated mastermix. FIG. 85 showstrans cleavage rates of different Cas12 variants upon complex formationwith a gRNA and a target sequence comprising different PAM sequences.PAM sequences and the sequences of the target and non-target strands areprovided in TABLE 29. Individual plots show trans cleavage rates foreach Cas12 variant, and each part illustrates the cleavage rate fortarget sequences comprising different PAM sequences. The pre-crRNA usedin each reaction wasGUUUCAAAGAUUAAAUAAUUUCUACUAAGUGUAGAUUCCUGCAGCAGAAAAUCAAAGACAAUGAAUAUUUCGGCGC (SEQ ID NO: 380). Trans cleavage activity wasmeasured as a function of cleavage rate. Variants including SEQ ID NO:11, SEQ ID NO: 575, SEQ ID NO: 581, SEQ ID NO: 587, and SEQ ID NO: 3exhibit transcleavage activity in the presence of a variety of PAMsequences.

In a second assay, each Cas12 variant was tested for sensitivity oftrans cleavage activity to base pair mismatches between the targetnucleic acid and the gRNA. To measure the tolerance of each Cas12variant to mismatches, single or double mismatches were introduced inthe first (“1MM”), fifth (“5MM”), tenth (“10MM”), fifteenth (“15MM”),and twentieth (“20MM”) nucleotide position after the PAM (TTTA (SEQ IDNO: 384)). Sequences of the mismatched strands are listed in TABLE 29.DETECTR trans cleavage assays were performed in the presence of activitybuffer (5 mM MgCl₂, 20 mM, Tris pH 7.5). Different dsDNA target nucleicacids with different base pair mismatched sequences were detected at afinal concentration of 100 nM. ssDNA reporters were present in eachreaction at a concentration of 50 nM. Target dsDNA was obtained byannealing complementary ssDNA primers at a ratio of 2:1 of non-targetstrand to target strand in hybridization buffer (50 mM NaCl, 1 mM TrispH 8.0, 0.1 mM EDTA), to ensure double-stranded DNA is being detectedinstead of single-stranded DNA. Pre-crRNA was ordered from Synthego. Theprotein of interest and the guide RNA were added to each tube andincubated for 20 minutes at 37° C. Each reaction contained 16 μL of theincubated mastermix. FIG. 87A shows a schematic of a Cas protein, gRNA,and target sequence complex comprising either a single base pairmismatch (top) or a double base pair mismatch (bottom) between the gRNAand the target sequence. FIG. 87B shows trans cleavage activity ofdifferent Cas12 variants upon complex formation with a gRNA and a targetsequence having either a single base pair mismatch (top) or a doublebase pair mismatch (bottom). The gRNA used in each reaction wasGUUUCAAAGAUUAAAUAAUUUCUACUAAGUGUAGAUUCCUGCAGCAGAAAAUCAAAGACAAUGAAUAUUUCGGCGC (SEQ ID NO: 380). Trans cleavage activity wasmeasured as a function of fluorescence. Most Cas12 variants were able totolerate both single and double mismatches starting from 15th and 20thposition with respect to the PAM sequence, but trans cleavage ratedecreased when a mismatch was introduced within the seed region(nucleotides at positions 1-10 of the spacer region at the PAM-proximalend). PAM sequences and the sequences of the target and non-targetstrands are provided in TABLE 29.

TABLE 29Substrate nucleic acid sequences for the target and non-target strandsSubstrates Non Target Strand Target Strand TTTT GCCCGCGGGATTTTTTCCTGCAGCGCGCCGAAATATTCATTGTCTTTG (SEQ ID NO: 381) AGAAAATCAAAGACAATGAATATATTTTCTGCTGCAGGAAAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 603)(SEQ ID NO: 604) TTTG GCCCGCGGGATTTTGTCCTGCAGC GCGCCGAAATATTCATTGTCTTTG(SEQ ID NO: 382) AGAAAATCAAAGACAATGAATAT ATTTTCTGCTGCAGGACAAAATCCTTCGGCGC CGCGGGC (SEQ ID NO: 605) (SEQ ID NO: 606) TTTCGCCCGCGGGATTTTCTCCTGCAGC GCGCCGAAATATTCATTGTCTTTG (SEQ ID NO: 383)AGAAAATCAAAGACAATGAATAT ATTTTCTGCTGCAGGAGAAAATCC TTCGGCGC CGCGGGC(SEQ ID NO: 607) (SEQ ID NO: 608) TTTA GCCCGCGGGATTTTATCCTGCAGCGCGCCGAAATATTCATTGTCTTTG (SEQ ID NO: 384) AGAAAATCAAAGACAATGAATATATTTTCTGCTGCAGGATAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 609)(SEQ ID NO: 610) TTGA GCCCGCGGGATTTGATCCTGCAGC GCGCCGAAATATTCATTGTCTTTG(SEQ ID NO: 385) AGAAAATCAAAGACAATGAATAT ATTTTCTGCTGCAGGATCAAATCCTTCGGCGC CGCGGGC (SEQ ID NO: 611) (SEQ ID NO: 612) TTCAGCCCGCGGGATTTCATCCTGCAGC GCGCCGAAATATTCATTGTCTTTG (SEQ ID NO: 386)AGAAAATCAAAGACAATGAATAT ATTTTCTGCTGCAGGATGAAATCC TTCGGCGC CGCGGGC(SEQ ID NO: 613) (SEQ ID NO: 614) TTAA GCCCGCGGGATTTAATCCTGCAGCGCGCCGAAATATTCATTGTCTTTG (SEQ ID NO: 387) AGAAAATCAAAGACAATGAATATATTTTCTGCTGCAGGATTAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 615)(SEQ ID NO: 616) TGTA GCCCGCGGGATTGTATCCTGCAGC GCGCCGAAATATTCATTGTCTTTG(SEQ ID NO: 388) AGAAAATCAAAGACAATGAATAT ATTTTCTGCTGCAGGATACAATCCTTCGGCGC CGCGGGC (SEQ ID NO: 617) (SEQ ID NO: 618) TCTAGCCCGCGGGATTCTATCCTGCAGC GCGCCGAAATATTCATTGTCTTTG (SEQ ID NO: 389)AGAAAATCAAAGACAATGAATAT ATTTTCTGCTGCAGGATAGAATCC TTCGGCGC CGCGGGC(SEQ ID NO: 619) (SEQ ID NO: 620) TATA GCCCGCGGGATTATATCCTGCAGCGCGCCGAAATATTCATTGTCTTTG (SEQ ID NO: 390) AGAAAATCAAAGACAATGAATATATTTTCTGCTGCAGGATATAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 621)(SEQ ID NO: 622) GTTA GCCCGCGGGATGTTATCCTGCAGC GCGCCGAAATATTCATTGTCTTTG(SEQ ID NO: 391) AGAAAATCAAAGACAATGAATAT ATTTTCTGCTGCAGGATAACATCCTTCGGCGC CGCGGGC (SEQ ID NO: 623) (SEQ ID NO: 624) CTTAGCCCGCGGGATCTTATCCTGCAGC GCGCCGAAATATTCATTGTCTTTG (SEQ ID NO: 392)AGAAAATCAAAGACAATGAATAT ATTTTCTGCTGCAGGATAAGATCC TTCGGCGC CGCGGGC(SEQ ID NO: 625) (SEQ ID NO: 626) ATTA GCCCGCGGGATATTATCCTGCAGCGCGCCGAAATATTCATTGTCTTTG (SEQ ID NO: 393) AGAAAATCAAAGACAATGAATATATTTTCTGCTGCAGGATAATATCC TTCGGCGC CGCGGGC (SEQ ID NO: 627)(SEQ ID NO: 628) 1MM GCCCGCGGGATTTTACCCTGCAGC GCGCCGAAATATTCATTGTCTTTGAGAAAATCAAAGACAATGAATAT ATTTTCTGCTGCAGGGTAAAATCC TTCGGCGC CGCGGGC(SEQ ID NO: 629) (SEQ ID NO: 630) 5MM GCCCGCGGGATTTTATCCTACAGCGCGCCGAAATATTCATTGTCTTTG AGAAAATCAAAGACAATGAATATATTTTCTGCTGTAGGATAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 631)(SEQ ID NO: 632) 10MM GCCCGCGGGATTTTATCCTGCAGC GCGCCGAAATATTCATTGTCTTTGGGAAAATCAAAGACAATGAATAT ATTTTCCGCTGCAGGATAAAATCC TTCGGCGC CGCGGGC(SEQ ID NO: 633) (SEQ ID NO: 634) 15MM GCCCGCGGGATTTTATCCTGCAGCGCGCCGAAATATTCATTGTCTTTG AGAAAGTCAAAGACAATGAATATACTTTCTGCTGCAGGATAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 635)(SEQ ID NO: 636) 20MM GCCCGCGGGATTTTATCCTGCAGC GCGCCGAAATATTCATTGTCTTTGAGAAAATCAAAGACAATGAATAT ATTTTCTGCTGCAGGATAAAATCC TTCGGCGC CGCGGGC(SEQ ID NO: 609) (SEQ ID NO: 610) 1-2MM GCCCGCGGGATTTTACTCTGCAGCGCGCCGAAATATTCATTGTCTTTG AGAAAATCAAAGACAATGAATATATTTTCTGCTGCAGAGTAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 637)(SEQ ID NO: 638) 3-4MM GCCCGCGGGATTTTATCTCGCAGC GCGCCGAAATATTCATTGTCTTTGAGAAAATCAAAGACAATGAATAT ATTTTCTGCTGCGAGATAAAATCC TTCGGCGC CGCGGGC(SEQ ID NO: 639) (SEQ ID NO: 640) 5-6MM GCCCGCGGGATTTTATCCTATAGCGCGCCGAAATATTCATTGTCTTTG AGAAAATCAAAGACAATGAATATATTTTCTGCTATAGGATAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 641)(SEQ ID NO: 642) 7-8MM GCCCGCGGGATTTTATCCTGCGAC GCGCCGAAATATTCATTGTCTTTGAGAAAATCAAAGACAATGAATAT ATTTTCTGTCGCAGGATAAAATCC TTCGGCGC CGCGGGC(SEQ ID NO: 643) (SEQ ID NO: 644) 9-10MM GCCCGCGGGATTTTATCCTGCAGTGCGCCGAAATATTCATTGTCTTTG GGAAAATCAAAGACAATGAATATATTTTCCACTGCAGGATAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 645)(SEQ ID NO: 646) 11-12MM GCCCGCGGGATTTTATCCTGCAGCGCGCCGAAATATTCATTGTCTTTG AAGAAATCAAAGACAATGAATATATTTCTTGCTGCAGGATAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 647)(SEQ ID NO: 648) 13-14MM GCCCGCGGGATTTTATCCTGCAGCGCGCCGAAATATTCATTGTCTTTG AGAGGATCAAAGACAATGAATATATCCTCTGCTGCAGGATAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 649)(SEQ ID NO: 650) 15-16MM GCCCGCGGGATTTTATCCTGCAGCGCGCCGAAATATTCATTGTCTTTG AGAAAGCCAAAGACAATGAATATGCTTTCTGCTGCAGGATAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 651)(SEQ ID NO: 652) 17-18MM GCCCGCGGGATTTTATCCTGCAGCGCGCCGAAATATTCATTGTCTTCA AGAAAATTGAAGACAATGAATATATTTTCTGCTGCAGGATAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 653)(SEQ ID NO: 654) 19-20MM GCCCGCGGGATTTTATCCTGCAGCGCGCCGAAATATTCATTGTCCCTG AGAAAATCAGGGACAATGAATATATTTTCTGCTGCAGGATAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 655)(SEQ ID NO: 656) 21-22MM GCCCGCGGGATTTTATCCTGCAGCGCGCCGAAATATTCATTGCTTTTG AGAAAATCAAAAGCAATGAATATATTTTCTGCTGCAGGATAAAATCC TTCGGCGC CGCGGGC (SEQ ID NO: 657)(SEQ ID NO: 658) dsDNA off GGCCAGTTTCATTTGAGCATTAAACCTGATGATGATAGCGTGCAGAA TGTCAAGTTCTGCACGCTATCATCCTTGACATTTAATGCTCAAATGAA ATCAGG ACTGGCC (SEQ ID NO: 659)(SEQ ID NO: 660) ssDNA off GCCCGCGGGATTTTCTCCTGCAGCAGAAAATCAAAGACAATGAATAT TTCGGCGC (SEQ ID NO: 607) ssDNA onGCGCCGAAATATTCATTGTCTTTG ATTTTCTGCTGCAGGAGAAAATCC CGCGGGC(SEQ ID NO: 608)

In a third assay, each Cas12 variant was tested for sensitivity of transcleavage activity to salt concentration. DETECTR trans cleavage assayswere performed in the presence of activity buffer (5 mM MgCl₂, 20 mM,Tris pH 7.5, 120 mM NaCl, and 1% glycerol). dsDNA target nucleic acidwas detected at a final concentration of 100 nM. ssDNA reporters werepresent in each reaction at a concentration of 50 nM. Target dsDNA wasobtained by annealing complementary ssDNA primers at a ratio of 2:1 ofnon-target strand to target strand in hybridization buffer (50 mM NaCl,1 mM Tris pH 8.0, 0.1 mM EDTA), to ensure double-stranded DNA is beingdetected instead of single-stranded DNA. Pre-crRNA was ordered fromSynthego. The protein of interest and the guide RNA were added to eachtube and incubated for 20 minutes at 37° C. Each reaction contained 16μL of the incubated mastermix. FIG. 88 shows trans cleavage activity ofdifferent Cas12 variants at different concentrations of NaCl. Most Cas12variants showed increased trans cleavage activity at low saltconcentrations. The gRNA used in each reaction wasGUUUCAAAGAUUAAAUAAUUUCUACUAAGUGUAGAUUCCUGCAGCAGAAAAUCAAAGACAAUGAAUAUUUCGGCGC (SEQ ID NO: 380). Trans cleavage activity wasmeasured as a function of fluorescence.

Example 35 Identification of Cas12 Variants with Pre-crRNA ProcessingActivity

This example describes the identification of Cas12 variants withpre-crRNA processing activity. Each Cas12 variant was tested forpre-crRNA processing activity. Processing of pre-crRNA was performed inthe presence of activity buffer (5 mM MgCl₂, 20 mM, Tris pH 7.5).Pre-crRNA cleavage assays were performed at 37° C. with 4-fold molarexcess of the Cas12 variant relative to synthesized crRNA (finalconcentrations of 100 nM and 50 nM, respectively). Unless otherwiseindicated, the reaction was quenched after 1 h with 2×RNA loading dye.Following quenching, reactions were denatured at 95° C. for 5 minutesbefore resolving by 15% denaturing PAGE in 0.5×TBE buffer. FIG. 89 showsurea PAGE gels of pre-crRNA processing activity of different Cas12variants in the presence (“+”) or absence (“−”) of a Cas protein. Bandsshown are RNA bands. Pre-crRNA processing activity was observed for mostCas12 variants, with different Cas12 variants processing at differentrates.

Example 36 Trans Cleavage Activity of Cas12 Variants in the Presence ofcrRNAs for Native Cas Proteins

This example describes trans cleavage activity of Cas12 variants in thepresence of crRNAs for native Cas proteins. Each Cas12 variant wastested for orthogonality of the corresponding crRNA to native Cas12proteins. Each Cas12 variant was incubated with different syntheticSynthego pre-crRNAs and trans cleavage activity was measured. Eachpre-crRNA differed in the repeat sequence. The repeat sequence in eachcrRNA was based on the repeat sequence found in each CRISPR locus ofCas12 proteins. Cas12 variants showed different trans cleavage activitywhen paired with different pre-crRNAs from different native Cas12proteins. DETECTR trans cleavage assays were performed in the presenceof activity buffer (5 mM MgCl₂, 20 mM, Tris pH 7.5). dsDNA targetnucleic acid was detected at a final concentration of 100 nM. ssDNAreporters were present in each reaction at a concentration of 50 nM.Target dsDNA was obtained by annealing complementary ssDNA primers at aratio of 2:1 of non-target strand to target strand in hybridizationbuffer (50 mM NaCl, 1 mM Tris pH 8.0, 0.1 mM EDTA), to ensuredouble-stranded DNA is being detected instead of single-stranded DNA.Pre-crRNA was ordered from Synthego. The protein of interest and theguide RNA were added to each tube and incubated for 20 minutes at 37° C.Each reaction contained 16 μL of the incubated mastermix. FIG. 90 showstrans cleavage activity of different Cas12 variants in the presence ofdifferent crRNAs based on the native crRNAs found in the CRISPR locusfor native Cas12 proteins. Trans cleavage activity was measured usingfluorescence. Using crRNAs with different variants showed differenttranscleavage activity, indicating that some Cas12 variants are morepromiscuous with respect to crRNA than others. Pre-crRNA sequences areprovided in TABLE 30. Sequence alignments of the repeat regions ofdifferent Cas12 variants aligned to the repeat sequence of LbCas12a (SEQID NO: 1) are shown in FIG. 92. Repeat sequences of the Cas12 variantscorrespond to SEQ ID NO: 508-SEQ ID NO: 520 and SEQ ID NO: 522-SEQ IDNO: 536. The repeat sequence of LbCas12a corresponds to SEQ ID NO: 521.The target sequence is set forth in SEQ ID NO: 610.

TABLE 30 Cas12 Ortholog Pre-crRNA Sequences Cas12 Ortholog SEQ ID NORepeat Sequence Spacer Sequence Pre-crRNA Sequence SEQ ID NO:GUCUAAACCUCAA UCCUGCAGCAGAA GUCUAAACCUCAAUGAAAAUUU 571 UGAAAAUUUCUAAAUCAAAGACA CUACUGUUUCCUGCAGCAGAAAA CUGUU (SEQ ID NO: 539) UCAAAGACA(SEQ ID NO: 508) (SEQ ID NO: 540) SEQ ID NO: CCUAAUAAUUUCU UCCUGCAGCAGAACCUAAUAAUUUCUACUGUUGUA 572 ACUGUUGUAGAU AAUCAAAGACAGAUUCCUGCAGCAGAAAAUCAA (SEQ ID NO: 509) (SEQ ID NO: 539) AGACA(SEQ ID NO: 541) SEQ ID NO: CUCGAAUACCUAU UCCUGCAGCAGAACUCGAAUACCUAUAUUAAAUUU 573 AUUAAAUUUCUA AAUCAAAGACACUACUUUUGUAGAUUCCUGCAGC CUUUUGUAGAU (SEQ ID NO: 539) AGAAAAUCAAAGACA(SEQ ID NO: 510) (SEQ ID NO: 542) SEQ ID NO: GUUUAAUAAACA UCCUGCAGCAGAAGUUUAAUAAACACUUAUAAUUU 575 CUUAUAAUUUCU AAUCAAAGACACUACUGUUGUAGAUUCCUGCAGC ACUGUUGUAGAU (SEQ ID NO: 539) AGAAAAUCAAAGACA(SEQ ID NO: 511) (SEQ ID NO: 543) SEQ ID NO: 11 GUUUGGUACCUUUCCUGCAGCAGAA GUUUGGUACCUUUAUUAAUUUC UAUUAAUUUCUA AAUCAAAGACAUACUAAGUGUAGAUUCCUGCAG CUAAGUGUAGAU (SEQ ID NO: 539) CAGAAAAUCAAAGACA(SEQ ID NO: 512) (SEQ ID NO: 544) SEQ ID NO: GUUGAGUAACCA UCCUGCAGCAGAAGUUGAGUAACCAUAAGAAAAUU 579 UAAGAAAAUUUC AAUCAAAGACAUCUACUGUGUAGAUUCCUGCAGC UACUGUGUAGAU (SEQ ID NO: 539) AGAAAAUCAAAGACA(SEQ ID NO: 513) (SEQ ID NO: 545) SEQ ID NO: GUUUAAUAAGUA UCCUGCAGCAGAAGUUUAAUAAGUAAUAAAUGUCU 580 AUAAAUGUCUAC AAUCAAAGACAACUGUAGUGUAGAUUCCUGCAG UGUAGUGUAGAU (SEQ ID NO: 539) CAGAAAAUCAAAGACA(SEQ ID NO: 514) (SEQ ID NO: 546) SEQ ID NO: AGUUAAAUAAUA UCCUGCAGCAGAAAGUUAAAUAAUAAGAAAGAAUU 581 AGAAAGAAUUUC AAUCAAAGACAUCUACUAGUGUAGAUUCCUGCA UACUAGUGUAGA (SEQ ID NO: 539) GCAGAAAAUCAAAGACA U(SEQ IDNO: 547) (SEQ ID NO: 515) SEQ ID NO: GUUAAGUAAUAU UCCUGCAGCAGAAGUUAAGUAAUAUAAAAGAAUUU 582 AAAAGAAUUUCU AAUCAAAGACACUACUAUUGUAGAUUCCUGCAGC ACUAUUGUAGAU (SEQ ID NO: 539) AGAAAAUCAAAGACA(SEQ ID NO: 516) (SEQ IDNO: 548) SEQ ID NO: GUUUGACCUACUA UCCUGCAGCAGAAGUUUGACCUACUAAUUAAAUUU 583 AUUAAAUUUCUA AAUCAAAGACACUACUGUUGUAGAUUCCUGCAGC CUGUUGUAGAU (SEQ ID NO: 539) AGAAAAUCAAAGACA(SEQ ID NO: 517) (SEQ ID NO: 549) SEQ ID NO: GGCUAAAAGUAA UCCUGCAGCAGAAGGCUAAAAGUAAUAAACAAUUU 584 and SEQ UAAACAAUUUCU AAUCAAAGACACUACUUUCGUAGAUUCCUGCAGC ID NO: 588 ACUUUCGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 518) (SEQ ID NO: 550) SEQ ID NO:GUCAAAUAGUAA UCCUGCAGCAGAA GUCAAAUAGUAACUAACAAUUU 587 CUAACAAUUUCUAAAUCAAAGACA CUACUUCGGUAGAUUCCUGCAGC CUUCGGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 519) (SEQ ID NO: 551) SEQ ID NO:AUCUACACAAAGU UCCUGCAGCAGAA AUCUACACAAAGUAGAGAUUCG 589 AGAGAUUCGAAUAAUCAAAGACA AAUGAGUUUUGACUCCUGCAGC GAGUUUUGAC (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 520) (SEQ ID NO: 552) LbCas12a GUUUCAAAGAUUUCCUGCAGCAGAA GUUUCAAAGAUUAAAUAAUUUC (SEQ ID NO: AAAUAAUUUCUAAAUCAAAGACA UACUAAGUGUAGAUUCCUGCAG 1) CUAAGUGUAGAU (SEQ ID NO: 539)CAGAAAAUCAAAGACA (SEQ ID NO: 521) (SEQ ID NO: 553) SEQ ID NO: 3GUCUAAGAACUU UCCUGCAGCAGAA GUCUAAGAACUUUAAAUAAUUU UAAAUAAUUUCUAAUCAAAGACA CUACUGUUGUAGAUUCCUGCAGC ACUGUUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 522) (SEQ ID NO: 554) SEQ ID NO:AUUUGAAAGCAU UCCUGCAGCAGAA AUUUGAAAGCAUCUUUUAAUUU 590 CUUUUAAUUUCUAAUCAAAGACA CUACUAUUGUAGAUUCCUGCAGC ACUAUUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 523) (SEQ ID NO: 555) SEQ ID NO:CUCUAAUAAGAG UCCUGCAGCAGAA CUCUAAUAAGAGAUAUGAAUUU 591 AUAUGAAUUUCUAAUCAAAGACA CUACUGUUGUAGAUUCCUGCAGC ACUGUUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 524) (SEQ ID NO: 556) Cas12 VariantCUCUACAACUGAU UCCUGCAGCAGAA CUCUACAACUGAUAAAGAAUUU AAAGAAUUUCUAAAUCAAAGACA CUACUUUUGUAGAUUCCUGCAGC CUUUUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 525) (SEQ ID NO: 557) Cas12 VariantCUCUAGCAGGCCU UCCUGCAGCAGAA CUCUAGCAGGCCUGGCAAAUUUC GGCAAAUUUCUACAAUCAAAGACA UACUGUUGUAGAUUCCUGCAGC UGUUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 526) (SEQ ID NO: 558) Cas12 VariantGCCAAAUACCUCU UCCUGCAGCAGAA GCCAAAUACCUCUAUAAAAUUUC AUAAAAUUUCUAAAUCAAAGACA UACUUUUGUAGAUUCCUGCAGC CUUUUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 527) (SEQ ID NO: 559) Cas12 VariantGCCAAGAACCUAU UCCUGCAGCAGAA GCCAAGAACCUAUAGAUAAUUU AGAUAAUUUCUAAAUCAAAGACA CUACUGUUGUAGAUUCCUGCAGC CUGUUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 528) (SEQ ID NO: 560) Cas12 VariantGGCUAUAAAGCU UCCUGCAGCAGAA GGCUAUAAAGCUUAUUUAAUUU UAUUUAAUUUCUAAUCAAAGACA CUACUAUUGUAGAUUCCUGCAGC ACUAUUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 529) (SEQ ID NO: 561) SEQ ID NO: 2GUCAAAAGACCUU UCCUGCAGCAGAA GUCAAAAGACCUUUUUAAUUUC UUUAAUUUCUACAAUCAAAGACA UACUCUUGUAGAUUCCUGCAGCA UCUUGUAGAU (SEQ ID NO: 539)GAAAAUCAAAGACA (SEQ ID NO: 530) (SEQ ID NO:562) Cas12 VariantGUCUAAAACUCAU UCCUGCAGCAGAA GUCUAAAACUCAUUCAGAAUUU UCAGAAUUUCUACAAUCAAAGACA CUACUAGUGUAGAUUCCUGCAGC UAGUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 531) (SEQ ID NO: 563) Cas12 VariantGUCUAACUACCUU UCCUGCAGCAGAA GUCUAACUACCUUUUAAUUUCU UUAAUUUCUACUAAUCAAAGACA ACUGUUUGUAGAUUCCUGCAGC GUUUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 532) (SEQ ID NO: 564) Cas12 VariantGUCUAUAAGACA UCCUGCAGCAGAA GUCUAUAAGACAUUUAUAAUUU UUUAUAAUUUCUAAUCAAAGACA CUACUAUUGUAGAUUCCUGCAGC ACUAUUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 533) (SEQ ID NO: 565) Cas12 VariantGUUUAAAACCACU UCCUGCAGCAGAA GUUUAAAACCACUUUAAAAUUU UUAAAAUUUCUAAAUCAAAGACA CUACUAUUGUAGAUUCCUGCAGC CUAUUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 534) (SEQ ID NO: 566) Cas12 VariantGUUUAAAAGUCC UCCUGCAGCAGAA GUUUAAAAGUCCUAUUGGAUUU UAUUGGAUUUCUAAUCAAAGACA CUACUUUUGUAGAUUCCUGCAGC ACUUUUGUAGAU (SEQ ID NO: 539)AGAAAAUCAAAGACA (SEQ ID NO: 535) (SEQ ID NO: 567) Cas12 VariantUGCUUAGAACAU UCCUGCAGCAGAA UGCUUAGAACAUUUAAAGAAUU UUAAAGAAUUUCAAUCAAAGACA UCUACUAUUGUAGAUUCCUGCA UACUAUUGUAGA (SEQ ID NO: 539)GCAGAAAAUCAAAGACA U (SEQ ID NO: 568) (SEQ ID NO: 536) Cas12 VariantUGCUUAGUACUU UCCUGCAGCAGAA UGCUUAGUACUUAUAAAGAAUU AUAAAGAAUUUCAAUCAAAGACA UCUACUAUUGUAGAUUCCUGCA UACUAUUGUAGA (SEQ ID NO: 539)GCAGAAAAUCAAAGACA U (SEQ ID NO: 569) (SEQ ID NO: 537) MAD7 GUUAAGUUAUAUUCCUGCAGCAGAA GUUAAGUUAUAUAGAAUAAUUU AGAAUAAUUUCU AAUCAAAGACACUACUUCCUGCAGCAGAAAAUCA ACU (SEQ ID NO: 539) AAGACA (SEQ ID NO: 538)(SEQ ID NO: 570)

Example 37 Cis Cleavage Activity of Cas12 Variants

This example describes the cis cleavage activity of Cas12 variants. Cis(target) cleavage assays were performed at 25° C. or 37° C. in activitybuffer (120 mM NaCl, 5 mM MgCl₂, 20 mM Tris pH 7.5, 1% glycerol). Cas12variant-crRNA complex formation was performed in activity buffer at amolar ratio of 1:1.25 protein to crRNA at 37° C. for 10 min. The ciscleavage target was a 1200 bp PCR product that contained the targetsequence at the 700th position. A restriction site for BamHI wasintroduced near the target sequence. Unless otherwise indicated, thefinal concentrations of protein, guide and targets were 100 nM, 125 nMand 15 nM, respectively, for all reactions. Cis cleavage assays wereperformed in the presence of activity buffer. Target dsDNA was detectedat a final concentration of 15 nM Target dsDNA was obtained by annealingcomplementary ssDNA primers at a ratio of 2:1 of non-target strand totarget strand in hybridization buffer (50 mM NaCl, 1 mM Tris pH 8.0, 0.1mM EDTA), to ensure double-stranded DNA is being detected instead ofsingle-stranded DNA. The protein of interest and the guide RNA wereadded to each tube and incubated for 20 minutes at 37° C. Each reactioncontained 16 μL of the incubated mastermix. Reactions were quenched with6× loading dye and resolved by pre-stained 2% agarose gel in 1×TAEbuffer. FIG. 91 shows cis cleavage activity of different Cas12 variantsafter incubation with a target nucleic acid sequence for 10 minutes.Cleavage with BamHI is shown as a cleavage positive control. DifferentCas12 variants demonstrate different rates of cis cleavage activity.

Example 38 Trans Cleavage Activity of a Cas12 Variant with DifferentgRNAs

This example describes the trans cleavage activity of a Cas12 variant ofSEQ ID NO: 11 with different gRNAs. A detection assay was performedusing gRNAs with either the repeat sequence of LbCas12a (SEQ ID NO: 1)or the repeat sequence of the Cas12 variant of SEQ ID NO: 11. Targetnucleic acid was detected at a final concentration of 1 nM or 0 nM(negative control).

FIG. 93 shows the results of an assay comparing DETECTR assay efficiencyfor a Cas12 variant of SEQ ID NO: 11 with two different gRNAs. The gRNAcontains either the LbCas12a repeat sequence (“gRNA #1,” SEQ ID NO: 423,UAAUUUCUACUAAGUGUAGAUUCAUCACGCAGCUCAUGCCC) or the Cas12 variant repeatsequence (“gRNA #2,” SEQ ID NO: 424,GUUUGGUACCUUUAUUAAUUUCUACUAAGUGUAGAUUCAUCACGCAGCUCAUGCC C). Thedetection reaction was performed at 37° C. for 30 minutes with 1 nMtarget DNA. A sample with 0 nM target DNA was tested as a negativecontrol. The results indicated that the Cas12 variant is compatible withthe gRNA corresponding to the repeat sequence of LbCas12a (“gRNA #1,”SEQ ID NO: 423). Results further indicated that the Cas12 variant showedincreased trans cleavage activity in the presence of the shorter gRNA(“gRNA #1,” SEQ ID NO: 423).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A composition comprising a programmable nuclease having at least 60%sequence identity to SEQ ID NO:11 and a non-naturally occurring guidenucleic acid.
 2. The composition of claim 1, further comprising adetector nucleic acid.
 3. The composition of claim 1, wherein theprogrammable nuclease recognizes a protospacer adjacent motif of5′-YYN-3′.
 4. The composition of claim 2, further comprising a targetnucleic acid, wherein when a region of said non-naturally occurringguide nucleic acid hybridizes to a portion of said target nucleic acidadjacent to a protospacer adjacent motif, said programmable nucleasecleaves detector nucleic acids at a rate of at least about 0.1 cleaveddetector nucleic acid molecules per minute.
 5. The composition of claim4, wherein the protospacer adjacent motif is 5′-YYN-3′.
 6. (canceled) 7.(canceled)
 8. (canceled)
 9. The composition of claim 1, wherein theprogrammable nuclease comprises three partial RuvC domains.
 10. Thecomposition of claim 1, wherein the programmable nuclease comprises aRuvC-I subdomain, a RuvC-II subdomain, and a RuvC-III subdomain. 11.(canceled)
 12. The composition of claim 1, wherein the programmablenuclease has at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 92%, at least 95%, at least 97%, or at least 99%sequence identity to SEQ ID NO:11.
 13. The composition of claim 1,wherein the programmable nuclease is SEQ ID NO:
 11. 14. (canceled) 15.(canceled)
 16. The composition of claim 1, wherein the compositionfurther comprises a buffer.
 17. The composition of claim 16, wherein thebuffer comprises a buffering agent, a salt, a crowding agent, adetergent, or any combination thereof.
 18. The composition of claim 17,wherein the buffering agent is at a concentration of from 5 mM to 100mM.
 19. (canceled)
 20. (canceled)
 21. The composition of claim 17,wherein the salt is from 5 mM to 100 mM.
 22. (canceled)
 23. Thecomposition of claim 17, wherein the crowding agent is from 0.5% (v/v)to 2% (v/v).
 24. (canceled)
 25. The composition of claim 17, wherein thedetergent is about 2% (v/v) or less.
 26. (canceled)
 27. The compositionof claim 17, wherein the buffering agent is HEPES.
 28. The compositionof claim 17, wherein the salt is potassium acetate, magnesium acetate,sodium chloride, magnesium chloride, or any combination thereof.
 29. Thecomposition of claim 17, wherein the crowding agent is glycerol.
 30. Thecomposition of claim 17, wherein the detergent is Tween, Triton-X, orany combination thereof.
 31. The composition of claim 17, wherein a pHof the composition is from 7 to
 8. 32. The composition of claim 16,wherein a pH of the buffer is approximately 7.5.
 33. The composition ofclaim 1, wherein the composition is at a temperature of from 25° C. to45° C.
 34. The composition of claim 1, wherein the programmable nucleaseexhibits catalytic activity at a temperature of from 25° C. to 45° C.35. The composition of claim 1, wherein the programmable nucleaseexhibits catalytic activity after heating the composition to atemperature of greater than 45° C. and restoring the temperature to from25° C. to 45° C.
 36. A method of assaying for a segment of a targetnucleic acid in a sample, the method comprising: contacting the sampleto: a reporter comprising a detector nucleic acid; and the compositionof claim 1, wherein the non-naturally occurring guide nucleic acidhybridizes to a segment of the target nucleic acid; and assaying for asignal produced by cleavage of the detector nucleic acid. 37.-183.(canceled)
 184. The method of claim 36, wherein the signal is presentprior to cleavage of the detector nucleic acid and changes upon cleavageof the detector nucleic acid or wherein the signal is absent prior tocleavage of the detector nucleic acid and is present upon cleavage ofthe detector nucleic acid.
 185. The method of claim 36, wherein thesignal is a calorimetric, potentiometric, amperometric, optical, orpiezo-electric signal.
 186. The method of claim 185, wherein the opticalsignal is a fluorescent or colorimetric signal.
 187. The method of claim36, further comprising amplifying the target nucleic acid.
 188. Thecomposition of claim 1, wherein the non-naturally occurring guidenucleic acid is a guide RNA.
 189. The composition of claim 188, whereinthe guide RNA comprises a sequence that is 80% homologous to SEQ IDNO:512.
 190. The composition of claim 1, wherein the non-naturallyoccurring guide nucleic acid has at least 60% sequence identity to SEQID NO:512.
 191. The composition of claim 1, wherein the non-naturallyoccurring guide nucleic acid comprises a crRNA.
 192. The composition ofclaim 1, wherein the non-naturally occurring guide nucleic acidcomprises RNA having at least 10 nucleotides that hybridize to a regionin a target nucleic acid adjacent to a protospacer adjacent motif. 193.The composition of claim 1, further comprising a reporter.
 194. Thecomposition of claim 193, wherein the reporter comprises a detectornucleic acid and a detection moiety.
 195. The composition of claim 194,wherein the detection moiety comprises a fluorescent moiety, a quenchingmoiety, a fluorescent dye, an infrared dye, an ultraviolet dye, afluorescence resonance energy transfer (FRET) pair, a polypeptide, abiotin, an avidin, a streptavidin, a polysaccharide, a polymer, or ananoparticle.
 196. The composition of claim 193, wherein the detectornucleic acid comprises DNA, RNA, or a combination thereof.
 197. Thecomposition of claim 1, further comprising an oligonucleotide primer oramplification reagent.
 198. The composition of claim 197, wherein theamplification reagent is selected from a recombinase, a single-strandedDNA binding (SSB) protein, a DNA polymerase, an RNA polymerase, adeoxynucleotide triphosphate, a nucleotide triphosphate, a reversetranscriptase, a helicase, a ligase, or any combination thereof.